SYSTEMS AND METHODS FOR MODIFYING THE FOLDING TRAJECTORY AND FACILITATING FOLDING OF POLYPEPTIDE CHAINS INTO NATIVE, NON-NATIVE, AND ARTIFICIAL CONFORMATIONS
20230197188 · 2023-06-22
Inventors
Cpc classification
International classification
Abstract
The present invention relates to peptide manipulation systems and methods for modifying the folding trajectory and facilitating the folding of a polypeptide chain into native, non-native, or artificial conformations. The invention comprises applying movement restriction(s) and/or directional rotation(s) to a plurality of locations along a peptide backbone. The applied movement restriction(s) and/or directional rotation(s) are sufficient to achieve twisting or other conformation changes of different portions of the peptide backbone and thereby modify peptide folding trajectory and facilitate peptide folding.
Claims
1. A system for modifying the folding trajectory and facilitating the folding of a polypeptide chain into native, non-native, or artificial conformations, the system comprising: at least one computing processor; and memory comprising instructions that, when executed by the at least one computing processor, enable the computing system to: apply at least one first movement restriction preventing rotation of at least one first portion of a peptide backbone; apply at least one first directional rotation to at least one second portion of the peptide backbone, wherein the first directional rotation and first movement restrictions are applied simultaneously; remove the applied first movement restriction and first directional rotation; apply at least one second movement restriction preventing rotation of at least one third portion of a peptide backbone; and apply at least one second directional rotation to at least one fourth portion of the peptide backbone, wherein the second directional rotation and second movement restriction are applied simultaneously.
2. The system of claim 1, wherein the applied movement restrictions and directional rotation cause at least one of twisting of the peptide backbone and a change in conformation of the polypeptide chain.
3. The system of claim 1, wherein applying the at least one first movement restriction comprises applying at least one of a force and a torque at a first location along the peptide backbone.
4. The system of claim 1, wherein applying the at least one first directional rotation comprises applying at least one of a force and a torque at a second location along the peptide backbone.
5. The system of claim 1, wherein the applying directional rotations and movement restrictions are performed in silico as part of a computer simulation.
6. The system of claim 1, wherein the applying directional rotations and movement restrictions are performed in vitro.
7. The system of claim 1, wherein the applying directional rotations comprises augmenting an established force field with the addition of a mechanical rotational force.
8. The system of claim 1, wherein the directional rotations are applied to at least one of an N-terminal amino acid, a C-terminal amino acid, and any other amino acid of the peptide backbone.
9. The system of claim 1, wherein the movement restrictions are applied to at least one of an N-terminal amino acid, a C-terminal amino acid, and any other amino acid of the peptide backbone.
10. The system of claim 1, wherein the applying directional rotations comprises applying at least one of electrical forces, magnetic forces, mechanical forces, and other forces.
11. The system of claim 1, wherein the applying movement restrictions comprises applying at least one of covalent and non-covalent binding characteristics to at least one of an amino acid within the peptide backbone, an atom within the peptide backbone, an atom in a peptide side chain, and an atom within an entity tightly bound to the peptide.
12. The system of claim 1, wherein the applying movement restrictions comprises modifying at least one of a temperature, a chemical composition, and a viscosity of a solvent in which the peptide is located.
13. A system for modifying the folding trajectory and facilitating the folding of a polypeptide chain into native, non-native, or artificial conformations, the system comprising: at least one peptide manipulator configured to physically manipulate the polypeptide chain, wherein the peptide manipulator is configured to: apply at least one first movement restriction preventing at least one of rotation or other movement of at least one first portion of a peptide backbone; apply at least one first directional rotation or other movement to at least one second portion of the peptide backbone, wherein the first directional rotation or other movement and first movement restrictions are applied simultaneously; remove the applied first movement restriction and first directional rotation or other movement; apply at least one second movement restriction preventing rotation or other movement of at least one third portion of a peptide backbone; and apply at least one second directional rotation or other movement to at least one fourth portion of the peptide backbone, wherein the second directional rotation or other movement and second movement restriction are applied simultaneously.
14. The system of claim 13, wherein the polypeptide manipulator is configured to apply movement restrictions and directional rotations or other movements using at least one of optical tweezers, magnetic tweezers, magnetic beads, electromagnets, piezoelectric devices, and piezoacoustic devices.
15. A method for modifying the folding trajectory and facilitating the folding of a polypeptide chain into native, non-native, or artificial conformations, the method comprising: applying at least one first movement restriction preventing rotation or other movement of at least one first portion of a peptide backbone; applying at least one first directional rotation to at least one second portion of the peptide backbone, wherein the first directional rotation or other movement and first movement restrictions are applied simultaneously; removing the applied first movement restriction and first directional rotation or other movement; applying at least one second movement restriction preventing rotation or other movement of at least one third portion of a peptide backbone; and applying at least one second directional rotation to at least one fourth portion of the peptide backbone, wherein the second directional rotation or other movement and second movement restriction are applied simultaneously.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] The accompanying drawings illustrate several embodiments and, together with the description, serve to explain the principles of the invention according to the embodiments. It will be appreciated by one skilled in the art that the particular arrangements illustrated in the drawings are merely exemplary and are not to be considered as limiting of the scope of the invention or the claims herein in any way.
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DETAILED DESCRIPTION
[0020] The inventive system and method (hereinafter sometimes referred to more simply as “system” or “method”) described herein significantly improves protein folding outcomes by manipulating polypeptide chains via direct manipulation. Specifically, the inventive system applies restraining and/or rotating forces at various locations along a polypeptide backbone to create at least one of twisting or other change of conformation of a polypeptide chain, performs various iterations of twisting or other change of conformation over time and provides a controlled release of the polypeptide chain to allow folding into native, non-native, and/or artificial conformations. The inventive system described herein provides an approach that incorporates new factors into the protein folding mechanism which have not previously been implemented into protein folding simulations or in vitro protein folding protocols.
[0021] One or more different embodiments may be described in the present application. The instant invention may be implemented with each embodiment individually or any combination of the embodiments disclosed. Further, for one or more of the embodiments described herein, numerous alternative arrangements may be described; it should be appreciated that these are presented for illustrative purposes only and are not limiting of the embodiments contained herein or the claims presented herein in any way. One or more of the arrangements may be widely applicable to numerous embodiments, as may be readily apparent from the disclosure. In general, arrangements are described in sufficient detail to enable those skilled in the art to practice one or more of the embodiments, and it should be appreciated that other arrangements may be utilized and that structural, logical, software, physical and other changes may be made without departing from the scope of the embodiments. Particular features of one or more of the embodiments described herein may be described with reference to one or more particular embodiments or figures that form a part of the present disclosure, and in which are shown, by way of illustration, specific arrangements of one or more of the aspects. It should be appreciated, however, that such features are not limited to usage in the one or more particular embodiments or figures with reference to which they are described. The present disclosure is neither a literal description of all arrangements of one or more of the embodiments nor a listing of features of one or more of the embodiments that must be present in all arrangements.
[0022] Headings of sections provided in this patent application and the title of this patent application are for convenience only and are not to be taken as limiting the disclosure in any way.
[0023] Devices that are in communication with each other need not be in continuous communication with each other, unless expressly specified otherwise. In addition, devices that are in communication with each other may communicate directly or indirectly through one or more communication means or intermediaries, logical or physical.
[0024] A description of an aspect with several components in communication with each other does not imply that all such components are required. To the contrary, a variety of optional components may be described to illustrate a wide variety of possible embodiments and in order to more fully illustrate one or more embodiments. Similarly, although process steps, method steps, algorithms or the like may be described in a sequential order, such processes, methods and algorithms may generally be configured to work in alternate orders, unless specifically stated to the contrary. In other words, any sequence or order of steps that may be described in this patent application does not, in and of itself, indicate a requirement that the steps be performed in that order. The steps of described processes may be performed in any order practical. Further, some steps may be performed simultaneously despite being described or implied as occurring non-simultaneously (e.g., because one step is described after the other step). Moreover, the illustration of a process by its depiction in a drawing does not imply that the illustrated process is exclusive of other variations and modifications thereto, does not imply that the illustrated process or any of its steps are necessary to one or more of the embodiments, and does not imply that the illustrated process is preferred. Also, steps are generally described once per aspect, but this does not mean they must occur once, or that they may only occur once each time a process, method, or algorithm is carried out or executed. Some steps may be omitted in some embodiments or some occurrences, or some steps may be executed more than once in a given aspect or occurrence.
[0025] When a single device or article is described herein, it will be readily apparent that more than one device or article may be used in place of a single device or article. Similarly, where more than one device or article is described herein, it will be readily apparent that a single device or article may be used in place of the more than one device or article.
[0026] The functionality or the features of a device may be alternatively embodied by one or more other devices that are not explicitly described as having such functionality or features. Thus, other embodiments need not include the device itself.
[0027] Techniques and mechanisms described or referenced herein will sometimes be described in singular form for clarity. However, it should be appreciated that particular embodiments may include multiple iterations of a technique or multiple instantiations of a mechanism unless noted otherwise. Process descriptions or blocks in figures should be understood as representing modules, segments, or portions of code which include one or more executable instructions for implementing specific logical functions or steps in the process which may be applied to in silico or in vitro protein manipulations. However, in vitro applications may include solely manual manipulations according to the process descriptions or block in figures, computer implemented or assisted manipulations via computer executable instructions (e.g. instructions for controlling a robotic manipulator) or combinations of manual and computer implemented manipulations. Alternate implementations are included within the scope of various embodiments in which, for example, functions may be executed out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, as would be understood by those having ordinary skill in the art.
[0028] The detailed description set forth below in connection with the appended drawings is intended as a description of various configurations and is not intended to represent the only configurations in which the concepts described herein may be practiced. The detailed description includes specific details for the purpose of providing a thorough understanding of various concepts. However, it will be apparent to those skilled in the art that these concepts may be practiced without these specific details. In some instances, well known structures and components are shown in block diagram form in order to avoid obscuring such concepts.
[0029]
[0030] User device(s) 101 may be used to communicate with a polypeptide manipulation and data processing system 102 and a datastore 103 via network 150. User device(s) 101 may function to allow a user to specify or select one or more polypeptide chains to be manipulated. In one embodiment, this may involve using the user device(s) 101 to enter one or more polypeptide sequences and transmitting such sequences to the polypeptide manipulation and data processing system 102. In one embodiment, the user device(s) 101 may be used to select one or more polypeptide chains from a list of polypeptide chains stored in a datastore 103 or stored in the polypeptide manipulation and data processing system 102. The list of polypeptide chains may include common natural protein sequences, and/or artificial sequences, and/or smaller polypeptide chains such as polypeptide chains which commonly make up larger polypeptide chains. User device(s) 101 may also be used to communicate settings and preferences for the polypeptide manipulation such as target intermediate or final conformations resulting from polypeptide manipulation, manipulation parameters such as forces and other parameters such as those discussed below with respect to
[0031] The polypeptide manipulation and data processing system 102 manipulates one or more polypeptide sequences based on a set of settings and parameters. These settings and parameters may be preset settings and parameters defined within the polypeptide manipulation and data processing system 102, settings and parameters received from user device(s) 101, and/or settings and parameters received from a datastore 103. The polypeptide manipulation and data processing system 102 may also receive an input specifying one or more polypeptide chains to be manipulated. This may involve receiving one or more particular polypeptide sequences from user device(s) 101 in the form of custom sequences and/or selection of one or more polypeptide sequences from a list as described above. In one embodiment, the polypeptide manipulation and data processing system 102 generates a polypeptide chain based on received input such as a received amino acid sequence.
[0032] In one embodiment, the polypeptide manipulation and data processing system 102 may be a server or cloud-based software application for performing polypeptide manipulation in silico. In one embodiment the polypeptide manipulation and data processing system 102 may take the form of a local application installed and running on a user device 101. In one embodiment, the polypeptide manipulation and data processing system 102 may include a variety of components for manipulating a polypeptide in vitro. Such components may include manipulators for applying mechanical forces, electrical forces, and/or magnetic forces. Some exemplary components may include one or more robotic arms and end effectors, components such as various solid phases for covalent, non-covalent and/or affinity protein immobilization, molecular tweezers, lasers, nanodevices, microfluidic devices, electromagnetic field generators, and the like which may be used separately or as one or more end effectors on robotic arms.
[0033] The datastore 103 may store manipulation settings and parameters used for a given polypeptide manipulation. The datastore 103 may store outcomes of manipulations such as resulting conformations, and videos of the manipulation and conformation changes. For example, the applied forces and their timing among other parameters such as, but not limited to, those discussed under
[0034] Polypeptide database 104 may comprise data associated with a plurality of polypeptide and/or protein structures, folding conformations, and/or folding trajectories. The polypeptide database 104 may comprise data associated with a plurality of known proteins and/or polypeptide chains. The polypeptide database 104 may be updated over time as new data associated with proteins and/or polypeptides becomes available, including but not limited to naturally occurring proteins, artificial proteins, misfolded protein conformations, and the like. In one aspect, data from the polypeptide database 104 is provided to polypeptide manipulation and data processing system 102 for use in computing outcome measures related to peptide backbone manipulation as described in more detail below.
[0035] Client device(s) 101 include, generally, a computer or computing device including functionality for communicating (e.g., remotely) over a network 150. Data may be collected from client devices 101, and data requests may be initiated from each client device 101. Client device(s) 101 may be a server, a desktop computer, a laptop computer, personal digital assistant (PDA), an in- or out-of-car navigation system, a smart phone or other cellular or mobile phone, or mobile gaming device, among other suitable computing devices. Client devices 101 may execute one or more client applications, such as a web browser (e.g., Microsoft Windows Internet Explorer, Mozilla Firefox, Apple Safari, Google Chrome, and Opera, etc.), or a dedicated application to submit user data, or to make prediction queries over a network 150.
[0036] In particular embodiments, each user device 101 may be an electronic device including hardware, software, or embedded logic components or a combination of two or more such components and capable of carrying out the appropriate functions implemented or supported by the user device 101. For example and without limitation, a user device 101 may be a desktop computer system, a notebook computer system, a netbook computer system, a handheld electronic device, or a mobile telephone. The present disclosure contemplates any user device 101. A user device 101 may enable a network user at the user device 101 to access network 150. A user device 101 may enable its user to communicate with other users at other user devices 101.
[0037] A user device 101 may have a web browser, such as MICROSOFT INTERNET EXPLORER, GOOGLE CHROME or MOZILLA FIREFOX, and may have one or more add-ons, plug-ins, or other extensions, such as TOOLBAR or YAHOO TOOLBAR. A user device 101 may enable a user to enter a Uniform Resource Locator (URL) or other address directing the web browser to a server, and the web browser may generate a Hyper Text Transfer Protocol (HTTP) request and communicate the HTTP request to server. The server may accept the HTTP request and communicate to the user device 101 one or more Hyper Text Markup Language (HTML) files responsive to the HTTP request. The user device 101 may render a web page based on the HTML files from server for presentation to the user. The present disclosure contemplates any suitable web page files. As an example and not by way of limitation, web pages may render from HTML files, Extensible Hyper Text Markup Language (XHTML) files, or Extensible Markup Language (XML) files, according to particular needs. Such pages may also execute scripts such as, for example and without limitation, those written in JAVASCRIPT, JAVA, MICROSOFT SILVERLIGHT, combinations of markup language and scripts such as AJAX (Asynchronous JAVASCRIPT and XML), and the like. Herein, reference to a web page encompasses one or more corresponding web page files (which a browser may use to render the web page) and vice versa, where appropriate.
[0038] The user device 101 may also include an application that is loaded onto the user device 101. The application obtains data from the network 150 and displays it to the user within the application 533 interface.
[0039] Exemplary user devices are illustrated in some of the subsequent figures provided herein. This disclosure contemplates any suitable number of user devices, including computing systems taking any suitable physical form. As example and not by way of limitation, computing systems may be an embedded computer system, a system-on-chip (SOC), a single-board computer system (SBC) (such as, for example, a computer-on-module (COM) or system-on-module (SOM)), a desktop computer system, a laptop or notebook computer system, an interactive kiosk, a mainframe, a mesh of computer systems, a mobile telephone, a personal digital assistant (PDA), a server, or a combination of two or more of these. Where appropriate, the computing system may include one or more computer systems; be unitary or distributed; span multiple locations; span multiple machines; or reside in a cloud, which may include one or more cloud components in one or more networks. Where appropriate, one or more computing systems may perform without substantial spatial or temporal limitation one or more steps of one or more methods described or illustrated herein. As an example, and not by way of limitation, one or more computing systems may perform in real time or in batch mode one or more steps of one or more methods described or illustrated herein. One or more computing system may perform at different times or at different locations one or more steps of one or more methods described or illustrated herein, where appropriate.
[0040] Network cloud 150 generally represents a network or collection of networks (such as the Internet or a corporate intranet, or a combination of both) over which the various components illustrated in
[0041] The network 150 connects the various systems and computing devices described or referenced herein. In particular embodiments, network 150 is an intranet, an extranet, a virtual private network (VPN), a local area network (LAN), a wireless LAN (WLAN), a wide area network (WAN), a metropolitan area network (MAN), a portion of the Internet, or another network 421 or a combination of two or more such networks 150. The present disclosure contemplates any suitable network 150.
[0042] One or more links couple one or more systems, engines or devices to the network 150. In particular embodiments, one or more links each includes one or more wired, wireless, or optical links. In particular embodiments, one or more links each includes an intranet, an extranet, a VPN, a LAN, a WLAN, a WAN, a MAN, a portion of the Internet, or another link or a combination of two or more such links. The present disclosure contemplates any suitable links coupling one or more systems, engines or devices to the network 150.
[0043] In particular embodiments, each system or engine may be a unitary server or may be a distributed server spanning multiple computers or multiple datacenters. Systems, engines, or modules may be of various types, such as, for example and without limitation, web server, news server, mail server, message server, advertising server, file server, application server, exchange server, database server, or proxy server. In particular embodiments, each system, engine or module may include hardware, software, or embedded logic components or a combination of two or more such components for carrying out the appropriate functionalities implemented or supported by their respective servers. For example, a web server is generally capable of hosting websites containing web pages or particular elements of web pages. More specifically, a web server may host HTML files or other file types, or may dynamically create or constitute files upon a request, and communicate them to clients devices or other devices in response to HTTP or other requests from clients devices or other devices. A mail server is generally capable of providing electronic mail services to various clients devices or other devices. A database server is generally capable of providing an interface for managing data stored in one or more data stores.
[0044] In particular embodiments, one or more data storages may be communicatively linked to one or more servers via one or more links. In particular embodiments, data storages may be used to store various types of information. In particular embodiments, the information stored in data storages may be organized according to specific data structures. In particular embodiments, each data storage may be a relational database. Particular embodiments may provide interfaces that enable servers or clients to manage, e.g., retrieve, modify, add, or delete, the information stored in data storage.
[0045] The system may also contain other subsystems and databases, which are not illustrated in
[0046]
[0047] User input interface 201 is operable to obtain user input associated with peptide manipulation. In one aspect user input comprises a set of parameters/settings to be used in peptide manipulation. Exemplary user input may comprise an indication of and/or settings or parameters associated with at least one movement restriction and/or at least one directional rotation to be applied to a peptide backbone. In one aspect, user input comprises at least one of a force to be applied (including, but not limited to, at least one of force type (e.g. mechanical, electrical, magnetic, etc.), force constant(s) and force magnitude to be used), a rate of directional rotation to be applied, a direction of rotation to be applied (e.g. clockwise or counter clockwise about the longitudinal axis of the peptide backbone), a duration of application of at least one of movement restriction(s) and directional rotation(s), and one or more locations at which each manipulation will be applied. Additional settings/parameters may be specified or determined from user input, such as those discussed below in association with
[0048] Simulation engine 204 is operable to execute computer simulations in order to apply movement restriction(s) and/or directional rotation(s) associated with the obtained user input. In one aspect, simulation engine 204 performs molecular dynamics simulations in association with the settings/parameters established based on the user input. Simulation engine 204 may execute a plurality of simulation processes associated with applying at least one of movement restriction(s) and/or directional rotation(s), such as that described in
[0049] Output control interface 206 is operable to output control signals to physical peptide manipulation components. In one aspect, the control signals are provided to components configured for performing in vitro peptide manipulation. Exemplary components include, but are not limited to one or more robotic arms and end effectors, components such as various solid phases for covalent, non-covalent and/or affinity protein immobilization, molecular tweezers, lasers, optical tweezers, magnetic tweezers, magnetic beads, electromagnets, piezoelectric devices, piezoacoustic devices, nanodevices, microfluidic devices, electromagnetic field generators, and the like.
[0050] Data acquisition interface 203 is operable to capture data associated with peptide manipulation. In one aspect, data acquisition interface 203 is operable to obtain data from an external system or component which acquires data associated with in vitro peptide manipulation. In one aspect, data acquisition interface 203 is operable to obtain, alone, or in combination/cooperation with simulation engine 204, snapshots or screenshots of peptide manipulation throughout the manipulation process. In one aspect, data acquisition interface obtains peptide manipulation data at least one of before, during, and after the application and/or removal of movement restriction(s) and/or directional rotation(s) applied to the peptide.
[0051] Polypeptide database interface 202 is operable to at least one of obtain information from a polypeptide database and provide information to a polypeptide database. In one aspect, polypeptide database interface 202 obtains information associated with known and/or expected folding trajectories and/or conformations associated with known peptide sequences (e.g. known proteins). In one aspect, polypeptide database interface 202 obtains information associated with known and/or expected folding trajectories and/or conformations associated with artificial peptide sequences (e.g. artificial proteins). In one aspect, polypeptide database interface 202 obtains information associated with rates or folding durations associated with at least one of known and unknown peptide chains.
[0052] Outcome measure engine 205 is operable to compute at least one outcome measure based on data obtained in association with at least one of data acquisition interface 203 and polypeptide database interface 202. In one aspect, outcome measure engine is operable to compute at least one of a folding trajectory of the polypeptide chain, a resulting folded conformation, a folding accuracy, the number or proportion of the native contacts formed by the amino acids, and a time to achieve the resulting folded conformation. In one aspect, the outcome measure engine 205 computes at least one of a measure of similarity and a measure of difference between at least one of the computed folding trajectory as compared to an expected folding trajectory and the computed resulting folded conformation as compared to an expected resulting folded conformation. In one aspect, the outcome measure engine 205 computes at least one of a measure of similarity and a measure of difference between at least one of the computed folding trajectory as compared to an expected folding trajectory and the computed resulting folded conformation as compared to an expected resulting folded conformation. In one aspect, the outcome measure engine 205 computes at least one of a measure of similarity and a measure of difference between an observed folding trajectory and an expected folding trajectory. In one aspect, the comparison of computed or observed folding trajectory and an expected folding trajectory can be used to identify if and when a peptide chain achieves or progresses towards a mis-folded conformation. In one aspect, the outcome measure engine 205 computes at least one of a measure of similarity and a measure of difference between at least one of a computed folding time and an expected folding time in order to determine a degree of augmentation resulting from the applied peptide manipulations.
[0053]
[0054] At step 301, the process comprises applying at least one first movement restriction to a peptide backbone 301. The movement restriction may comprise applying at least one of a force and a torque to the peptide backbone. In one aspect, applying at least one first movement restriction may comprise applying a plurality of movement restrictions at one or more locations along the peptide backbone. The movement restriction may comprise a restriction to at least one of prevent rotation of the peptide backbone and limit rotation of the peptide backbone. In one aspect, limiting rotation may comprise limiting at least one of an amount of rotation and a rate of rotation of the peptide backbone. In one aspect, movement restriction(s) is/are applied via at least one of an established force field, electrical force(s), magnetic force(s), mechanical force(s), covalent and/or non-covalent binding forces or characteristics, and other force(s). In one aspect, applying at least one first movement restriction may comprise applying at least one force to an amino acid, where the at least one force is of sufficient magnitude to restrict movement of at least one of the amino acid and portions of the peptide backbone in proximity to the amino acid. In one aspect, applying at least one first movement restriction may comprise applying at least one force to a plurality of amino acids, where the at least one force is of sufficient magnitude to restrict movement of at least the plurality of amino acid and portions of the peptide backbone in proximity to the plurality of amino acids. In one aspect, the movement restriction(s) are applied to at least one of an N-terminal amino acid, a C-terminal amino acid, and any other amino acid of the peptide backbone. In one aspect, applying movement restriction(s) comprises modifying at least one of a temperature, a chemical composition, and a viscosity of a solvent in which the peptide is located.
[0055] At step 302, the process comprises applying at least one first directional rotation to the peptide backbone. In one aspect, the direction of rotation comprises at least one of clockwise or counterclockwise rotation about the longitudinal axis of the peptide backbone. The directional rotation may comprise applying at least one of a force and a torque to the peptide backbone. In one aspect, applying at least one first directional rotation may comprise applying a plurality of directional rotations at one or more locations along the peptide backbone. In one aspect, directional rotation(s) is/are applied via at least one of an established force field, electrical force(s), magnetic force(s), mechanical force(s), covalent and/or non-covalent binding forces or characteristics, and other force(s). In one aspect, applying at least one first directional rotation may comprise applying at least one force to an amino acid, where the at least one force is of sufficient magnitude to rotate at least one of the amino acid and portions of the peptide backbone in proximity to the amino acid. In one aspect, applying at least one first directional rotation may comprise applying at least one force to a plurality of amino acids, where the at least one force is of sufficient magnitude to rotate at least the plurality of amino acid and portions of the peptide backbone in proximity to the plurality of amino acids. In one aspect, the directional rotation(s) are applied to at least one of an N-terminal amino acid, a C-terminal amino acid, and any other amino acid of the peptide backbone. In one aspect, the directional rotation(s) is/are applied using an enforced rotation method. In one aspect, the directional rotation(s) is/are applied over a threshold time period. In one aspect, the directional rotation(s) is/are applied using a flexible axis approach with a defined reference rotation rate.
[0056] At step 303, the process comprises removing the applied first movement restriction(s) and directional rotation(s). In one aspect, removing the restrictions and/or rotations may involve removing all applied manipulations simultaneously. In one aspect, removing may involve removing the applied manipulations in a particular order. In one aspect, removing may comprise at least one of removing individual applied manipulations one at a time, removing groups of applied manipulations one at a time. In one aspect, removing the applied movement restriction(s) and directional rotation(s) comprises a controlled release of the constraints on the peptide backbone, such as a gradual or stepwise decrease in the forces applied in order to impose the restrictions and/or rotations. In one aspect, the controlled release may involve removing all or some applied manipulations with additional application of manipulations at one or more of the same locations as previously applied forces and/or additional application of forces at one or more different locations as previously applied constraints. In one embodiment, the controlled release may involve maintaining one or more applied manipulation at the end of this step.
[0057] At step 304, the process comprises applying at least one second movement restriction to the peptide backbone. This may be performed in the same fashion as applying the first movement restriction as described in step 301 above. The second movement restriction(s) may be applied at the same and/or different locations as the at least one first movement restriction(s) applied in step 301.
[0058] At step 305, the process comprises applying at least one second directional rotation to the peptide backbone. This may be performed in the same fashion as applying the first directional rotation as described in step 302 above. The second directional rotation(s) may be applied at the same and/or different locations as the at least one first directional rotation(s) applied in step 302.
[0059] In one aspect, the combined application of movement restriction(s) and directional rotation(s) are performed in such a way as to induce at least one of a twisting of the peptide backbone and a change of conformation of the peptide backbone. In one aspect, the twisting or other change of conformation of the peptide backbone serves to modify the folding trajectory and facilitate the folding of a polypeptide chain into native, non-native, and/or artificial conformations. In one aspect, the application of movement restriction(s) and directional rotation(s) augment peptide folding by accelerating folding by a defined factor (e.g. reduce the time needed to achieve a folded, stable conformation). In one aspect, the application of movement restriction(s) and/or directional rotation(s) are adjusted and/or modified as necessary in order to achieve a desired or predefined twisting or other conformation change of the peptide backbone.
[0060] In one aspect, the above application of movement restriction(s) and directional rotation(s) may comprise performing the applications in silico as part of a computer simulation. For example, molecular dynamics simulation software may be used to apply the movement restriction(s) and directional rotation(s). In one aspect, the computer simulations are performed using at least one of a constant temperature, a constant pressure, and a defined force constant. In one aspect, the movement restriction(s) and directional rotation(s) are applied in vitro. In one aspect, in vitro application of movement restriction(s) and directional rotation(s) are performed using at least one of optical tweezers, magnetic tweezers, magnetic beads, electromagnets, piezoelectric devices, and piezoacoustic devices. In one aspect, the process is used to produce at least one artificial protein having at least one of a native and a non-native folding conformation.
[0061] In one aspect, the process may further comprise capturing images before, during, and/or after at least one of the application and removal of movement restriction(s) and directional rotation(s). In one aspect, capturing images comprises obtaining snapshots or screenshots via a computer simulation based application and removal of the movement restriction(s) and directional rotation(s).
[0062] In one aspect, the process may further comprise computing at least one outcome measure associated with peptide manipulation. In one aspect, the outcome measure(s) are associated with at least one of folding trajectory, resulting folded conformation, folding accuracy, the number or proportion of the native contacts, and a time to achieve the resulting folding conformation. In one aspect, the folding accuracy comprises at least one of a measure of similarity and a measure of difference between at least one of the computed folding trajectory as compared to an expected folding trajectory and the computed resulting folded conformation as compared to an expected resulting folded conformation. In one aspect, the process may further comprise identifying at least one of at least one native folding confirmation achieved, at least one non-native folding conformation achieved, at least one stable folding conformation achieved, at least one non-stable folding conformation achieved, at least one functional conformation achieved, at least one intermediary confirmation achieved (e.g. an alpha helix), and at least one misfolded conformation achieved.
[0063] Generally, the techniques disclosed herein may be implemented on hardware or a combination of software and hardware. For example, they may be implemented in an operating system kernel, in a separate user process, in a library package bound into network applications, on a specially constructed machine, on an application-specific integrated circuit (ASIC), or on a network interface card.
[0064] Software/hardware hybrid implementations of at least some of the embodiments disclosed herein may be implemented on a programmable network-resident machine (which should be understood to include intermittently connected network-aware machines) selectively activated or reconfigured by a computer program stored in memory. Such network devices may have multiple network interfaces that may be configured or designed to utilize different types of network communication protocols. A general architecture for some of these machines may be described herein in order to illustrate one or more exemplary means by which a given unit of functionality may be implemented. According to specific embodiments, at least some of the features or functionalities of the various embodiments disclosed herein may be implemented on one or more general-purpose computers associated with one or more networks, such as for example an end-user computer system, a client computer, a network server or other server system, a mobile computing device (e.g., tablet computing device, mobile phone, smartphone, laptop, or other appropriate computing device), a consumer electronic device, a music player, or any other suitable electronic device, router, switch, or other suitable device, or any combination thereof. In at least some embodiments, at least some of the features or functionalities of the various embodiments disclosed herein may be implemented in one or more virtualized computing environments (e.g., network computing clouds, virtual machines hosted on one or more physical computing machines, or other appropriate virtual environments). Any of the above mentioned systems, units, modules, engines, controllers, components or the like may be and/or comprise hardware and/or software as described herein. For example, the polypeptide manipulation and data processing system 102 and subcomponents thereof may be and/or comprise computing hardware and/or software as described herein in association with
[0065] Referring now to
[0066] In one aspect, computing device 10 includes one or more central processing units (CPU) 12, one or more interfaces 15, and one or more busses 14 (such as a peripheral component interconnect (PCI) bus). When acting under the control of appropriate software or firmware, CPU 12 may be responsible for implementing specific functions associated with the functions of a specifically configured computing device or machine. For example, in at least one aspect, a computing device 10 may be configured or designed to function as a server system utilizing CPU 12, local memory 11 and/or remote memory 16, and interface(s) 15. In at least one aspect, CPU 12 may be caused to perform one or more of the different types of functions and/or operations under the control of software modules or components, which for example, may include an operating system and any appropriate applications software, drivers, and the like.
[0067] CPU 12 may include one or more processors 13 such as, for example, a processor from one of the Intel, ARM, Qualcomm, and AMD families of microprocessors. In some embodiments, processors 13 may include specially designed hardware such as application-specific integrated circuits (ASICs), electrically erasable programmable read-only memories (EEPROMs), field-programmable gate arrays (FPGAs), and so forth, for controlling operations of computing device 10. In a particular aspect, a local memory 11 (such as non-volatile random-access memory (RAM) and/or read-only memory (ROM), including for example one or more levels of cached memory) may also form part of CPU 12. However, there are many different ways in which memory may be coupled to system 10. Memory 11 may be used for a variety of purposes such as, for example, caching and/or storing data, programming instructions, and the like. It should be further appreciated that CPU 12 may be one of a variety of system-on-a-chip (SOC) type hardware that may include additional hardware such as memory or graphics processing chips, such as a QUALCOMM SNAPDRAGON™ or SAMSUNG EXYNOS™ CPU as are becoming increasingly common in the art, such as for use in mobile devices or integrated devices.
[0068] As used herein, the term “processor” is not limited merely to those integrated circuits referred to in the art as a processor, a mobile processor, or a microprocessor, but broadly refers to a microcontroller, a microcomputer, a programmable logic controller, an application-specific integrated circuit, and any other programmable circuit.
[0069] In one aspect, interfaces 15 are provided as network interface cards (NICs). Generally, NICs control the sending and receiving of data packets over a computer network; other types of interfaces 15 may for example support other peripherals used with computing device 10. Among the interfaces that may be provided are Ethernet interfaces, frame relay interfaces, cable interfaces, DSL interfaces, token ring interfaces, graphics interfaces, and the like. In addition, various types of interfaces may be provided such as, for example, universal serial bus (USB), Serial, Ethernet, FIREWIRE™, THUNDERBOLT™, PCI, parallel, radio frequency (RF), BLUETOOTH™, near-field communications (e.g., using near-field magnetics), 802.11 (WiFi), frame relay, TCP/IP, ISDN, fast Ethernet interfaces, Gigabit Ethernet interfaces, Serial ATA (SATA) or external SATA (ESATA) interfaces, high-definition multimedia interface (HDMI), digital visual interface (DVI), analog or digital audio interfaces, asynchronous transfer mode (ATM) interfaces, high-speed serial interface (HSSI) interfaces, Point of Sale (POS) interfaces, fiber data distributed interfaces (FDDIs), and the like. Generally, such interfaces 15 may include physical ports appropriate for communication with appropriate media. In some cases, they may also include an independent processor (such as a dedicated audio or video processor, as is common in the art for high-fidelity A/V hardware interfaces) and, in some instances, volatile and/or non-volatile memory (e.g., RAM).
[0070] Although the system shown in
[0071] Regardless of network device configuration, the system of an aspect may employ one or more memories or memory modules (such as, for example, remote memory block 16 and local memory 11) configured to store data, program instructions for the general-purpose network operations, or other information relating to the functionality of the embodiments described herein (or any combinations of the above). Program instructions may control execution of or comprise an operating system and/or one or more applications, for example. Memory 16 or memories 11, 16 may also be configured to store data structures, configuration data, encryption data, historical system operations information, or any other specific or generic non-program information described herein.
[0072] Because such information and program instructions may be employed to implement one or more systems or methods described herein, at least some network device embodiments may include nontransitory machine-readable storage media, which, for example, may be configured or designed to store program instructions, state information, and the like for performing various operations described herein. Examples of such nontransitory machine-readable storage media include, but are not limited to, magnetic media such as hard disks, floppy disks, and magnetic tape; optical media such as CD-ROM disks; magneto-optical media such as optical disks, and hardware devices that are specially configured to store and perform program instructions, such as read-only memory devices (ROM), flash memory (as is common in mobile devices and integrated systems), solid state drives (SSD) and “hybrid SSD” storage drives that may combine physical components of solid state and hard disk drives in a single hardware device (as are becoming increasingly common in the art with regard to personal computers), memristor memory, random access memory (RAM), and the like. It should be appreciated that such storage means may be integral and non-removable (such as RAM hardware modules that may be soldered onto a motherboard or otherwise integrated into an electronic device), or they may be removable such as swappable flash memory modules (such as “thumb drives” or other removable media designed for rapidly exchanging physical storage devices), “hot-swappable” hard disk drives or solid state drives, removable optical storage discs, or other such removable media, and that such integral and removable storage media may be utilized interchangeably. Examples of program instructions include both object code, such as may be produced by a compiler, machine code, such as may be produced by an assembler or a linker, byte code, such as may be generated by for example a JAVA™ compiler and may be executed using a Java virtual machine or equivalent, or files containing higher level code that may be executed by the computer using an interpreter (for example, scripts written in Python, Perl, Ruby, Groovy, or any other scripting language).
[0073] In some embodiments, systems may be implemented on a standalone computing system. Referring now to
[0074] In some embodiments, systems may be implemented on a distributed computing network, such as one having any number of clients and/or servers. Referring now to
[0075] In addition, in some embodiments, servers 32 may call external services 37 when needed to obtain additional information, or to refer to additional data concerning a particular call. Communications with external services 37 may take place, for example, via one or more networks 31. In various embodiments, external services 37 may comprise web-enabled services or functionality related to or installed on the hardware device itself. For example, in one aspect where client applications are implemented on a smartphone or other electronic device, client applications may obtain information stored in a server system 32 in the cloud or on an external service 37 deployed on one or more of a particular enterprise's or user's premises.
[0076] In some embodiments, clients 33 or servers 32 (or both) may make use of one or more specialized services or appliances that may be deployed locally or remotely across one or more networks 31. For example, one or more databases 34 may be used or referred to by one or more embodiments. It should be understood by one having ordinary skill in the art that databases 34 may be arranged in a wide variety of architectures and using a wide variety of data access and manipulation means. For example, in various embodiments one or more databases 34 may comprise a relational database system using a structured query language (SQL), while others may comprise an alternative data storage technology such as those referred to in the art as “NoSQL” (for example, HADOOP CASSANDRA™, GOOGLE BIGTABLE™, and so forth). In some embodiments, variant database architectures such as column-oriented databases, in-memory databases, clustered databases, distributed databases, or even flat file data repositories may be used according to the aspect. It will be appreciated by one having ordinary skill in the art that any combination of known or future database technologies may be used as appropriate, unless a specific database technology or a specific arrangement of components is specified for a particular aspect described herein. Moreover, it should be appreciated that the term “database” as used herein may refer to a physical database machine, a cluster of machines acting as a single database system, or a logical database within an overall database management system. Unless a specific meaning is specified for a given use of the term “database”, it should be construed to mean any of these senses of the word, all of which are understood as a plain meaning of the term “database” by those having ordinary skill in the art.
[0077] Similarly, some embodiments may make use of one or more security systems 36 and configuration systems 35. Security and configuration management are common information technology (IT) and web functions, and some amount of each are generally associated with any IT or web systems. It should be understood by one having ordinary skill in the art that any configuration or security subsystems known in the art now or in the future may be used in conjunction with embodiments without limitation, unless a specific security 36 or configuration system 35 or approach is specifically required by the description of any specific aspect.
[0078]
[0079] In various embodiments, functionality for implementing systems or methods of various embodiments may be distributed among any number of client and/or server components. For example, various software modules may be implemented for performing various functions in connection with the system of any particular aspect, and such modules may be variously implemented to run on server and/or client components.
[0080] The skilled person will be aware of a range of possible modifications of the various embodiments described above. Accordingly, the present invention is defined by the claims and their equivalents.
[0081] A first example implementation and corresponding results of the above inventive concepts is included below.
[0082] Once they are synthesized in a living cell, the majority of proteins rapidly attain their distinctive biologically active three-dimensional structures, called native conformations. These conformations are robustly achieved in vivo via a folding process that involves interactions of the folding chain with molecular chaperones and other maturation factors. The native conformations of many proteins can be predicted using modern artificial intelligence methods, but the folding process leading to those native conformations usually cannot be reproduced either in silico or in vitro, except for those of a few relatively short polypeptides. Knowledge of the intermediates in the folding pathways and the mechanisms that enable them is essential for determining the points of intervention at which folding and misfolding processes can be altered.
[0083] The current dominant model of protein folding was prompted by early observations that some small proteins are able to fold in vitro into their native conformations spontaneously, in isolation from other proteins or cellular components. These observations gave rise to the thermodynamic hypothesis of protein folding, which in turn led to the development of the physical model that describes protein folding as a thermodynamically favorable, unassisted process. In a more recent, refined form, this model includes the description of a rugged funnel-shaped energy landscape, in which the various unfolded, unstructured conformations occupy the high-free-energy brim of the funnel. As the polypeptide chains fold, they sample conformations with progressively decreasing Gibbs free energy until they reach the native conformation, which is presumed to occupy the global thermodynamic minimum at the bottom of the funnel. The sampling of conformations during the folding process is assumed to occur via random thermal motions. The driving force of protein folding is assumed to be the decrease in free energy to the global minimum.
[0084] In summary, the current general physical model of protein folding describes a process that occurs in a closed system in the absence of external sources of energy. It assumes that folding starts from a random, unstructured conformation and proceeds unassisted, with no apparent requirement for the folding chain to interact with other proteins or macromolecular cellular components.
[0085] As the first step in exploring the feasibility of a protein folding machine capable of facilitating the attainment of native structure by mechanical manipulation of the peptide backbone, we performed molecular dynamics simulations augmented by application of torsion to the peptide backbones. During the simulations, the C-termini of various polypeptides were mechanically rotated either clockwise or counterclockwise, while the motions of their N-termini were restricted. We compared the trajectories of both types of simulations with the folding of the same peptides without the application of torque. In our experiments, directional rotation of the C-terminal amino acids with simultaneous limitation of the movements of the N-termini indeed facilitated the formation of native structures in five diverse alpha-helical peptides.
[0086] The initial stretched structures of peptides (shown in
[0087] For all simulations, we used the ff14SB force field with the TIP3P water model and ion parameters modified by Joung and Cheatham. Electrostatic interactions were calculated using particle-mesh Ewald (PME) summation with a Fourier grid spacing of 0.135 nm. For non-bonded Coulomb and Lennard-Jones interactions, 1 nm cutoff was used. We constrained the hydrogen bonds with the LINCS algorithm and used a 2-fs integration time step.
[0088] To exert an external mechanical torque to the C-termini of the peptides, we applied the enforced rotation. To this end, we restrained the positions of the O and N atoms of the C-terminal alanine to keep it aligned with the X-axis, about which the rotation was applied. The restraints with a force constant of 10000 kJ/mol*nm.sup.2 were applied only for the YZ-plane, so the C-terminal amino acid could move along the X-axis. In addition, we restrained the O atom of the C-terminal amino acid in the X direction with a force constant of 5 kJ/mol*nm.sup.2 and N and Ca atoms of the N-terminal alanine with a force constant 10000 kJ/mol*nm.sup.2 in all directions. The C-terminal amino acid was rotated using a flexible axis approach (Vflex2) with a reference rotation rate of 60 degrees/ps and a force constant of 1500 kJ/mol*nm.sup.2.
[0089] The GROMACS package version 2020.236 was used for all simulations and trajectory analyses. The simulations were carried out on CUDA-enabled GPUs with Turing architecture, running Ubuntu 18.04. For visualization of protein structures and trajectories, the programs ICM-Pro 3.924 and VMD 1.9.337 were used.
[0090]
[0091] The experiments were run on five peptides that are known to adopt α-helical conformations in their folded form (
[0092] For all five peptides, folding was observed when the rotation force was applied to the C-terminal amino acid in the clockwise direction (
[0093] Thus, the directional rotation of the C-terminal amino acid with simultaneous restriction of the movements of the N-terminal amino acid facilitated the formation of native structures in five diverse α-helical peptides, confirming that such constraints can have significant consequences for folding dynamics. Strikingly, application of mechanical force accelerated the folding of P4, a fragment of an on-pathway folding intermediate of the well-studied villin headpiece domain HP35, which is one of the fastest-folding protein domains known. The several-fold increase in the rate of P4 folding that was achieved in our experiments seems to suggest that the postulated “physical limit of folding” of HP35 as a whole could be overcome by a protein folding machine. The other four peptides in our experiments likewise attained their α-helical structure in the presence of the rotating force, but did not reach their native conformations when allowed to fold unassisted, even though we ran the control unassisted simulations for ˜10 times longer than the simulations that included the application of the external force (
[0094] A second example implementation and corresponding results of the above inventive concepts is included below.
[0095] We next conducted all-atom molecular dynamics simulations of three small protein domains, whose folding from an extended state was augmented by the application of rotational force to the C-terminal amino acid, while the movement of the N-terminal amino acid was restrained. The simulation protocol was changed to apply the backbone rotation and movement restriction for a limited time at the start of simulation. As will be shown below, this transient application of a mechanical force to the peptide is sufficient to accelerate, by at least an order of magnitude, the folding of three protein domains with different structure to their native or nearly-native structures. Our in silico experiments show that the native or native-like fold may be attained more readily when the movements of the polypeptide are biased by external forces and constraints.
[0096] In one aspect backbone twisting (or other conformation change) was applied for a defined time at the start of the simulation, and then the remainder of the simulation continued without restraints. The force was applied transiently for the first 250 ns of simulation, with the reference rate 0.36 degree/ps and a force constant 10000 kJ mol-1 nm-2. As in earlier experiments, we applied the rotation against the direction of an α-helix. Control simulations were performed in the boxes with mostly the same properties as in the rotation-augmented runs, but without restraints or manipulations of the polypeptide at any point. To monitor the protein folding process, we used several metrics throughout the simulation, collecting snapshots every 100 ps. At the whole-domain level, we measured the RMSD distance from the known native three-dimensional structure of the folded domain and the fraction of native contacts formed by all amino acids within the domain. At the residue level, we tested whether the residue was incorporated into a correct element of the secondary structure and whether it formed a native contact.
[0097] For our simulations, we selected protein domains whose folding in vitro and in molecular dynamics simulations has been extensively studied. Two of those domains are proteins designed for rapid folding and stability. They are Tpr-cage domain (PDB ID 2jof, 20 amino acids), which consists of a helix-coil-turn motif and a short proline tail that packs against the helix, and BBA domain (PDB ID 1fme, 28 amino acids), a derivative of a Zn finger that is stable in a metal-free form, consisting of two β-strands followed by an α-helix. Both peptides were placed in their simulation boxes in the stretched conformations, and both peptides rapidly folded to their native structures in the presence of directional rotation. The details of the folding process are presented in
[0098] The molecular dynamics (MD) simulations were carried out using the GROMACS-2020.4 package with CHARMM36m force field (July 2020 release). The stretched structures of proteins were produced with the ICM-Pro program. The proteins were aligned along the X-axis and solvated in triclinic boxes with TIP3P water and potassium and chloride ions. The system was then minimized with the steepest descent algorithm and equilibrated with NVT/NPT ensembles for 1 ns at each equilibration cycle. The MD simulations were performed at a constant temperature of 300 K with the v-rescale thermostat and a time constant of 0.1 ps. The pressure was coupled at 1 bar with Parrinello-Rahman barostat using a time constant of 2 ps. Electrostatic forces were calculated using the particle-mesh Ewald method with a Fourier grid spacing of 0.16 nm. Short-range Van der Waals interactions were force-switched off from 0.8 to 1.2 nm. The hydrogen-containing bonds were constrained using the LINCS algorithm, and 2 fs integration time step was used. Snapshots were collected every 100 ps.
[0099] The rotation of C-terminal amino acid was performed using the enforced rotation method implemented in Gromacs as in our previous study. The rotation was applied during the first 250 ns to the C-terminal amino acid using the flexible axis approach (Vflex2) with reference rotation rate 0.36 degree/ps (1 rotation per 1 ns) and force constant 10000 kJ*mol-1*nm-2, and continued without restraints and without applied rotation thereafter. Most control simulations were performed in the same box and with the same parameters, but without any initial restraint or rotation; see text for more discussion of some control runs for Trp cage and BBA domains.
[0100] The RMS distances from the database-deposited native structure were calculated in Gromacs. Acquisition of secondary structure and the fraction of native contacts were assessed using VMD software. VMD was also used for trajectory visualization. Native contacts for an amino acid were considered to be formed if that amino acid has the same contacts as in the reference X-ray structure within a cutoff 8 Å. The fraction of native contacts (Q) was calculated as Q=N/Nall where N is the number of the native contacts at a given time and Nall is the number of contacts in the reference X-ray structure.
[0101] A more detailed view of the folding process is provided by the analysis of secondary structure and native contacts at the residue level. Trp-cage domain and BBA domain each contain a helical segment in their native structure—respectively, in the N-terminal and C-terminal region of the polypeptide chain. As can be seen in
[0102] In the unassisted control simulations, the two polypeptides formed neither helices nor other elements of the secondary or tertiary structure. Short substructures with helix-like or strand-like properties could be seen, but these states were occupied only transiently and/or were located in the regions that have different type of structure in the native form of the protein (
[0103] For each target, the summaries of independent steered simulations can be compared side by side at the residue level, providing valuable visual information about the folding pathways (
[0104] We next studied folding of a naturally occurring protein domain that is somewhat longer than Trp-cage and BBA domains. The fragment of the N-terminal domain of ribosomal protein L9 from Geobacillus stearothermophilus (NTL9-39; PDB ID 2hba, 39 amino acids, K 12M mutant), consists of a β-sheet formed by three anti-parallel β-strands, and an α-helix that is located between strands 2 and 3 along the peptide chain and is packed against one edge of the sheet. The folding of the 39-residue form, which does not include the long α-helix connecting the N-terminal and C-terminal domains of the L9 protein, has been studied in vitro and in silico before. We simulated folding of this protein in the same way as for the other two domains, except that the simulation box was 10.5 nm long in the X axis direction, and therefore the starting peptide was not fully stretched (calculated fully-stretched length 12.8 nm). The results of our folding simulations for this domain are shown in
[0105] In the rotation-assisted simulations, NTL 9 folded into stable and compact form quickly, within 1-1.5 μs (
[0106] The structure that was formed in the rotation-assisted runs remained stable for the entire simulation time, but displayed interesting local differences from the known native conformation of NTL9. Focusing on the run 2, the majority of the fold, i.e., the helix and strands 1 and 2 that directly interact with it, were all formed nearly perfectly. The third beta β-strand was also observed, completing the three-stranded β-sheet similar to the wild-type fold. The examination of the snapshots in the course of the simulation, however, revealed a dynamic transition between two distinct conformations. One conformation was nearly identical to the native structure (
[0107] The results of the above applied manipulations indicate this novel approach to peptide manipulation successfully modified folding trajectories of proteins. In one aspect, folding was accelerated approximately ten fold or more as compared to what is known in the art. The inventive approach also provides a means for detecting misfolding and the characteristics associated with misfolding occurrences and/or detection of folding trajectory inflection points where folding towards non-native, but stable conformations occurs.
ADDITIONAL CONSIDERATIONS
[0108] As used herein any reference to “one embodiment” or “an embodiment” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.
[0109] Some embodiments may be described using the expression “coupled” and “connected” along with their derivatives. For example, some embodiments may be described using the term “coupled” to indicate that two or more elements are in direct physical or electrical contact. The term “coupled,” however, may also mean that two or more elements are not in direct contact with each other, but yet still co-operate or interact with each other. The embodiments are not limited in this context.
[0110] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and Bis false (or not present), A is false (or not present) and Bis true (or present), and both A and B are true (or present).
[0111] In addition, use of the “a” or “an” are employed to describe elements and components of the embodiments herein. This is done merely for convenience and to give a general sense of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.
[0112] Upon reading this disclosure, those of skill in the art will appreciate still additional alternative structural and functional designs for a system and a process for creating an interactive message through the disclosed principles herein. Thus, while particular embodiments and applications have been illustrated and described, it is to be understood that the disclosed embodiments are not limited to the precise construction and components disclosed herein. Various apparent modifications, changes and variations may be made in the arrangement, operation and details of the method and apparatus disclosed herein without departing from the spirit and scope defined in the appended claims.