@article {152150, title = {Sequence of the SARS-CoV-2 Spike Transmembrane Domain Encodes Conformational Dynamics.}, journal = {J Phys Chem B}, volume = {129}, year = {2025}, month = {2024 Dec 18}, pages = {194-209}, abstract = {

The homotrimeric SARS-CoV-2 spike protein enables viral infection by undergoing a large conformational transition, which facilitates the fusion of the viral envelope with the host cell membrane. The spike protein is anchored to the SARS-CoV-2 envelope by its transmembrane domain (TMD), composed of three TM helices, each contributed by one of the protomers of spike. Although the TMD is known to be important for viral fusion, whether it is a passive anchor of the spike or actively promotes fusion remains unknown. Specifically, it is unclear if the TMD and its dynamics facilitate the prefusion to postfusion conformational transition of the spike. Here, we computationally study the dynamics and self-assembly of the SARS-CoV-2 spike TMD in homogeneous POPC and cholesterol containing membranes. Atomistic simulations of a long TM helix-containing protomer segment show that the membrane-embedded segment bobs, tilts and gains and loses helicity, locally thinning the membrane. Coarse-grained multimerization simulations using representative TM helix structures from the atomistic simulations exhibit diverse trimer populations whose architecture depends on the structure of the TM helix protomer. While a symmetric conformation reflects the symmetry of the resting spike, an asymmetric TMD conformation could promote membrane fusion through the stabilization of a fusion intermediate. Together, our simulations demonstrate that the sequence and length of the SARS-CoV-2 spike TM segment make it inherently dynamic, that trimerization does not abrogate these dynamics and that the various observed TMD conformations may enable viral fusion.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.4c05270}, author = {Lall, Sahil and Balaram, Padmanabhan and Mathew, M K and Gosavi, Shachi} } @article {125198, title = {Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity.}, journal = {Front Mol Biosci}, volume = {10}, year = {2023}, month = {2023}, pages = {1021733}, abstract = {

Kinetic stability, defined as the rate of protein unfolding, is central to determining the functional lifetime of proteins, both in nature and in wide-ranging medical and biotechnological applications. Further, high kinetic stability is generally correlated with high resistance against chemical and thermal denaturation, as well as proteolytic degradation. Despite its significance, specific mechanisms governing kinetic stability remain largely unknown, and few studies address the rational design of kinetic stability. Here, we describe a method for designing protein kinetic stability that uses protein long-range order, absolute contact order, and simulated free energy barriers of unfolding to quantitatively analyze and predict unfolding kinetics. We analyze two β-trefoil proteins: hisactophilin, a quasi-three-fold symmetric natural protein with moderate stability, and ThreeFoil, a designed three-fold symmetric protein with extremely high kinetic stability. The quantitative analysis identifies marked differences in long-range interactions across the protein hydrophobic cores that partially account for the differences in kinetic stability. Swapping the core interactions of ThreeFoil into hisactophilin increases kinetic stability with close agreement between predicted and experimentally measured unfolding rates. These results demonstrate the predictive power of readily applied measures of protein topology for altering kinetic stability and recommend core engineering as a tractable target for rationally designing kinetic stability that may be widely applicable.

}, issn = {2296-889X}, doi = {10.3389/fmolb.2023.1021733}, author = {Anderson, Delaney M and Jayanthi, Lakshmi P and Gosavi, Shachi and Meiering, Elizabeth M} } @article {123071, title = {Potential Self-Peptide Inhibitors of the SARS-CoV-2 Main Protease.}, journal = {J Phys Chem B}, year = {2023}, month = {2023 Jan 23}, abstract = {

The SARS-CoV-2 main protease (M) plays an essential role in viral replication, cleaving viral polyproteins into functional proteins. This makes M an important drug target. M consists of an N-terminal catalytic domain and a C-terminal α-helical domain (MC). Previous studies have shown that peptides derived from a given protein sequence (self-peptides) can affect the folding and, in turn, the function of that protein. Since the SARS-CoV-1 MC is known to stabilize its M and regulate its function, we hypothesized that SARS-CoV-2 MC-derived self-peptides may modulate the folding and the function of SARS-CoV-2 M. To test this, we studied the folding of MC in the presence of various self-peptides using coarse-grained structure-based models and molecular dynamics simulations. In these simulations of MC and one self-peptide, we found that two self-peptides, the α1-helix and the loop between α4 and α5 (loop4), could replace the equivalent native sequences in the MC structure. Replacement of either sequence in full-length M should, in principle, be able to perturb M function albeit through different mechanisms. Some general principles for the rational design of self-peptide inhibitors emerge: The simulations show that prefolded self-peptides are more likely to replace native sequences than those which do not possess structure. Additionally, the α1-helix self-peptide is kinetically stable and once inserted rarely exchanges with the native α1-helix, while the loop4 self-peptide is easily replaced by the native loop4, making it less useful for modulating function. In summary, a prefolded α1-derived peptide should be able to inhibit SARS-CoV-2 M function.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.2c05917}, author = {Banerjee, Arkadeep and Gosavi, Shachi} } @article {119607, title = {The diversity of protein-protein interaction interfaces within T=3 icosahedral viral capsids}, journal = {Front Mol Biosci. }, volume = {9:967877}, year = {2022}, month = {10/2022}, abstract = {

Some non-enveloped virus capsids assemble from multiple copies of a single type of coat-protein (CP). The comparative energetics of the diverse CP-CP interfaces present in such capsids likely govern virus assembly-disassembly mechanisms. The T = 3 icosahedral capsids comprise 180 CP copies arranged about two-, three-, five- and six-fold axes of (quasi-)rotation symmetry. Structurally diverse CPs can assemble into T = 3 capsids. Specifically, the Leviviridae CPs are structurally distinct from the Bromoviridae, Tombusviridae and Tymoviridae CPs which fold into the classic "jelly-roll" fold. However, capsids from across the four families are known to disassemble into dimers. To understand whether the overall symmetry of the capsid or the structural details of the CP determine virus assembly-disassembly mechanisms, we analyze the different CP-CP interfaces that occur in the four virus families. Previous work studied protein homodimer interfaces using interface size (relative to the monomer) and hydrophobicity. Here, we analyze all CP-CP interfaces using these two parameters and find that the dimerization interface (present between two CPs congruent through a two-fold axis of rotation) has a larger relative size in the Leviviridae than in the other viruses. The relative sizes of the other Leviviridae interfaces and all the jelly-roll interfaces are similar. However, the dimerization interfaces across families have slightly higher hydrophobicity, potentially making them stronger than other interfaces. Finally, although the CP-monomers of the jelly-roll viruses are structurally similar, differences in their dimerization interfaces leads to varied dimer flexibility. Overall, differences in CP-structures may induce different modes of swelling and assembly-disassembly in the T = 3 viruses.

}, doi = {10.3389/fmolb.2022.967877}, author = {Prakash, DL and Gosavi, Shachi} } @article {115012, title = {A Method for Assessing the Robustness of Protein Structures by Randomizing Packing Interactions.}, journal = {Front Mol Biosci}, volume = {9}, year = {2022}, month = {2022}, pages = {849272}, abstract = {

Many single-domain proteins are not only stable and water-soluble, but they also populate few to no intermediates during folding. This reduces interactions between partially folded proteins, misfolding, and aggregation, and makes the proteins tractable in biotechnological applications. Natural proteins fold thus, not necessarily only because their structures are well-suited for folding, but because their sequences optimize packing and fit their structures well. In contrast, folding experiments on the designed Top7 suggest that it populates several intermediates. Additionally, in protein design, where sequences are designed for natural and new non-natural structures, tens of sequences still need to be tested before success is achieved. Both these issues may be caused by the specific scaffolds used in design, i.e., some protein scaffolds may be more tolerant to packing perturbations and varied sequences. Here, we report a computational method for assessing the response of protein structures to packing perturbations. We then benchmark this method using designed proteins and find that it can identify scaffolds whose folding gets disrupted upon perturbing packing, leading to the population of intermediates. The method can also isolate regions of both natural and designed scaffolds that are sensitive to such perturbations and identify contacts which when present can rescue folding. Overall, this method can be used to identify protein scaffolds that are more amenable to whole protein design as well as to identify protein regions which are sensitive to perturbations and where further mutations should be avoided during protein engineering.

}, issn = {2296-889X}, doi = {10.3389/fmolb.2022.849272}, author = {Yadahalli, Shilpa and Jayanthi, Lakshmi P and Gosavi, Shachi} } @article {102046, title = {Rational Design of Protein-Specific Folding Modifiers.}, journal = {J Am Chem Soc}, year = {2021}, month = {2021 Nov 01}, abstract = {

Protein-folding can go wrong and , with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, F{\"o}rster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 {\textpm} 5\%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

}, issn = {1520-5126}, doi = {10.1021/jacs.1c09611}, author = {Das, Anirban and Yadav, Anju and Gupta, Mona and R, Purushotham and Terse, Vishram L and Vishvakarma, Vicky and Singh, Sameer and Nandi, Tathagata and Banerjee, Arkadeep and Mandal, Kalyaneswar and Gosavi, Shachi and Das, Ranabir and Ainavarapu, Sri Rama Koti and Maiti, Sudipta} } @article {97223, title = {Understanding the Folding Mediated Assembly of the Bacteriophage MS2 Coat Protein Dimers.}, journal = {J Phys Chem B}, year = {2021}, month = {2021 Aug 02}, abstract = {

The capsids of RNA viruses such as MS2 are great models for studying protein self-assembly because they are made almost entirely of multiple copies of a single coat protein (CP). Although CP is the minimal repeating unit of the capsid, previous studies have shown that CP exists as a homodimer (CP2) even in an acid-disassembled system, indicating that CP2 is an obligate dimer. Here, we investigate the molecular basis of this obligate dimerization using coarse-grained structure-based models and molecular dynamics simulations. We find that, unlike monomeric proteins of similar size, CP populates a single partially folded ensemble whose "foldedness" is sensitive to denaturing conditions. In contrast, CP2 folds similarly to single-domain proteins populating only the folded and the unfolded ensembles, separated by a prominent folding free energy barrier. Several intramonomer contacts form early, but the CP2 folding barrier is crossed only when the intermonomer contacts are made. A dissection of the structure of CP2 through mutant folding simulations shows that the folding barrier arises both from the topology of CP and the interface contacts of CP2. Together, our results show that CP2 is an obligate dimer because of kinetic stability, that is, dimerization induces a folding barrier and that makes it difficult for proteins in the dimer minimum to partially unfold and access the monomeric state without completely unfolding. We discuss the advantages of this obligate dimerization in the context of dimer design and virus stability.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.1c03928}, author = {Lalwani, DP and Gosavi, Shachi} } @article {80848, title = {Protein energy landscape exploration with structure-based models.}, journal = {Curr Opin Struct Biol}, volume = {64}, year = {2020}, month = {2020 Aug 11}, pages = {145-151}, abstract = {

Exploring the multi-dimensional energy landscape of a large protein in detail is a computational challenge. Such investigations may include analysis of multiple folding pathways, rate constants for important conformational transitions, locating intermediate states populated during folding, estimating energetic and entropic barriers that separate populated basins, and visualising a high-dimensional surface. The complexity of the landscape can be simplified through coarse-grained structure-based models (SBMs). These widely used coarse-grained representations of proteins provide a minimalist approximation to the free energy landscape, which subsumes the folding behaviour of many single-domain proteins. Here we describe the combination of SBMs with discrete path sampling (DPS), and show how this approach can provide details of the landscape and folding pathways. Combining SBMs and DPS provides an efficient framework for sampling the protein free energy landscape and for calculating various kinetic and thermodynamic quantities.

}, issn = {1879-033X}, doi = {10.1016/j.sbi.2020.07.003}, author = {Neelamraju, Sridhar and Wales, David J and Gosavi, Shachi} } @article {67834, title = {Successes and challenges in simulating the folding of large proteins.}, journal = {J Biol Chem}, volume = {295}, year = {2020}, month = {2019 Nov 11}, pages = {15-33}, abstract = {

Computational simulations of protein folding can be used to interpret experimental folding results, to design new folding experiments and to test the effects of mutations and small molecules on folding. However, while major experimental and computational progress has been made in understanding how small proteins fold, research on larger, multi-domain proteins, which comprise the majority of proteins, is less advanced. Specifically, large proteins often fold via long-lived partially folded intermediates, whose structures, potentially toxic oligomerization and interactions with cellular chaperones remain poorly understood. Molecular dynamics (MD) based folding simulations that rely on knowledge of the native structure can provide critical, detailed information on folding free energy landscapes, intermediates and pathways. Further, increases in computational power and methodological advances have made folding simulations of large proteins practical and valuable. Here, using serpins that inhibit proteases as an example, we review native-centric methods for simulating the folding of large proteins. These synergistic approaches range from Go and related structure-based models (SBMs) that can predict the effects of the native structure on folding to all-atom-based methods that include side chain chemistry and can predict how disease-associated mutations may impact folding. The application of these computational approaches to serpins and other large proteins highlights the successes and limitations of current computational methods and underscores how computational results can be used to inform experiments. These powerful simulation approaches in combination with experiments can provide unique insights into how large proteins fold and misfold expanding our ability to predict and manipulate protein folding.

}, issn = {1083-351X}, doi = {10.1074/jbc.REV119.006794}, author = {Gershenson, Anne and Gosavi, Shachi and Faccioli, Pietro and Wintrode, Patrick L} } @article {50995, title = {A five-residue motif for the design of domain swapping in proteins.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 01 28}, pages = {452}, abstract = {

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue {\textquoteright}cystatin motif{\textquoteright} (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two β-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse β-hairpin proteins.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08295-x}, author = {Nandwani, Neha and Surana, Parag and Negi, Hitendra and Mascarenhas, Nahren M and Udgaonkar, Jayant B and Das, Ranabir and Gosavi, Shachi} } @article {55790, title = {Go-Kit: A Tool To Enable Energy Landscape Exploration of Proteins.}, journal = {J Chem Inf Model}, year = {2019}, month = {2019 Apr 12}, abstract = {

Coarse-grained Go̅-like models, based on the principle of minimal frustration, provide valuable insight into fundamental questions in the field of protein folding and dynamics. In conjunction with commonly used molecular dynamics (MD) simulations, energy landscape exploration methods like discrete path sampling (DPS) with Go̅-like models can provide quantitative details of the thermodynamics and kinetics of proteins. Here we present Go-kit, a software that facilitates the setup of MD and DPS simulations of several flavors of Go̅-like models. Go-kit is designed for use with MD (GROMACS) and DPS (PATHSAMPLE) simulation engines that are open source. The Go-kit code is written in python2.7 and is also open source. A case study for the ribosomal protein S6 is discussed to illustrate the utility of the software, which is available at https://github.com/gokit1/gokit .

}, issn = {1549-960X}, doi = {10.1021/acs.jcim.9b00007}, author = {Neelamraju, Sridhar and Wales, David J and Gosavi, Shachi} } @article {49277, title = {Energy Landscape of the Designed Protein Top7.}, journal = {J Phys Chem B}, year = {2018}, month = {2018 Nov 29}, abstract = {

To fold on biologically relevant timescales, proteins have evolved funnelled energy landscapes with minimal energetic trapping. However, the polymeric nature of proteins and the spatial arrangement of secondary structural elements can create topological traps and slow folding. It is challenging to identify, visualise and quantify such topological trapping. Designed proteins have not had the benefit of evolution and it has been hypothesised that de novo designed protein topologies may therefore feature more topological trapping. Structure-based models (SBMs) are inherently funnelled and remove most energetic trapping and can thus be used to isolate the effect of protein topology on the folding energy landscape. Here, we compare properties of the potential energy landscapes (PEL) of Top7, a designed protein with a topology unknown in nature, with those of S6, a naturally occurring ribosomal protein of similar size and topology. Possible kinetic traps and the energetic barriers separating them from the native state are elucidated. We find that even with an SBM, the PEL of the designed protein is more frustrated than that of the natural protein. We then quantify the effect of adding non-native hydrophobic interactions and coarse-grained side-chains through a frustration density parameter. A clear increase in frustration is observed on including side-chains, whereas adding hydrophobic interactions leads to a narrowing of the funnel and a decrease in complexity. The most likely (un)folding routes for all models are derived through the construction of "probability contact maps". The ability to quantitatively understand and optimise the organisation of the PEL for designed proteins may enable us to sculpt structure-seeking landscapes, mimicking the effect of evolution.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.8b08499}, author = {Neelamraju, Sridhar and Gosavi, Shachi and Wales, David J} } @article {27273, title = {Exploring the effects of sparse restraints on protein structure prediction.}, journal = {Proteins}, volume = {86}, year = {2018}, month = {02/2018}, pages = {248-262}, abstract = {

One of the main barriers to accurate computational protein structure prediction is searching the vast space of protein conformations. Distance restraints or inter-residue contacts have been used to reduce this search space, easing the discovery of the correct folded state. It has been suggested that about one contact for every twelve residues may be sufficient to predict structure at fold level accuracy. Here, we use coarse-grained structure-based models in conjunction with molecular dynamics simulations to examine this empirical prediction. We generate sparse contact maps for 15 proteins of varying sequence lengths and topologies and find that given perfect secondary-structural information, a small fraction of the native contact map (5-10\%) suffices to fold proteins to their correct native states. We also find that different sparse maps are not equivalent and we make several observations about the type of maps that are successful at such structure prediction. Long range contacts are found to encode more information than shorter range ones, especially for α and αβ-proteins. However, this distinction reduces for β-proteins. Choosing contacts that are a consensus from successful maps gives predictive sparse maps as does choosing contacts that are well spread out over the protein structure. Additionally, the folding of proteins can also be used to choose predictive sparse maps. Overall, we conclude that structure-based models can be used to understand the efficacy of structure-prediction restraints and could, in future, be tuned to include specific force-field interactions, secondary structure errors and noise in the sparse maps. This article is protected by copyright. All rights reserved.

}, issn = {1097-0134}, doi = {10.1002/prot.25438}, author = {Mandalaparthy, Varun and Sanaboyana, Venkata Ramana and Rafalia, Hitesh and Gosavi, Shachi} } @article {29624, title = {On the folding of a structurally complex protein to its metastable active state.}, journal = {Proc Natl Acad Sci U S A}, year = {2018}, month = {2018 Jan 17}, abstract = {

For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a β-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a β-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state.

}, issn = {1091-6490}, doi = {10.1073/pnas.1708173115}, author = {Giri Rao, V V Hemanth and Gosavi, Shachi} } @article {29626, title = {Intrinsic Disorder in a Well-Folded Globular Protein.}, journal = {J Phys Chem B}, year = {2018}, month = {2018 Feb 01}, abstract = {

The folded structure of the heterodimeric sweet protein monellin mimics single-chain proteins with topology β1-α1-β2-β3-β4-β5 (chain A: β3-β4-β5; chain B: β1-α1-β2). Furthermore, like naturally occurring single-chain proteins of a similar size, monellin folds cooperatively with no detectable intermediates. However, the two monellin chains, A and B, are marginally structured in isolation and fold only upon binding to each other. Thus, monellin presents a unique opportunity to understand the design of intrinsically disordered proteins that fold upon binding. Here, we study the folding of a single-chain variant of monellin (scMn) using simulations of an all heavy-atom structure-based model. These simulations can explain mechanistic details derived from scMn experiments performed using several different structural probes. scMn folds cooperatively in our structure-based simulations, as is also seen in experiments. We find that structure formation near the transition-state ensemble of scMn is not uniformly distributed but is localized to a hairpin-like structure which contains one strand from each chain (β2, β3). Thus, the sequence and the underlying energetics of heterodimeric monellin promote the early formation of the interchain interface (β2-β3). By studying computational scMn mutants whose "interchain" interactions are deleted, we infer that this energy distribution allows the two protein chains to remain largely disordered when this interface is not folded. From these results, we suggest that cutting the protein backbone of a globular protein between residues which lie within its folding nucleus may be one way to construct two disordered fragments which fold upon binding.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.7b12546}, author = {Mascarenhas, Nahren Manuel and Terse, Vishram L and Gosavi, Shachi} } @article {20316, title = {Amino-acid composition after loop deletion drives domain swapping.}, journal = {Protein Sci}, volume = {26}, year = {2017}, month = {10/2017}, chapter = {1994}, abstract = {

Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain-swapped oligomer over the monomer. While it is known that the deletion of loops between β-strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop-deletion mutants of the non-domain-swapping protein monellin, frame-shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.3237}, author = {Nandwani, Neha and Surana, Parag and Udgaonkar, Jayant B and Das, Ranabir and Gosavi, Shachi} } @article {26283, title = {Characterizing the conformational landscape of MDM2-binding p53 peptides using Molecular Dynamics simulations.}, journal = {Sci Rep}, volume = {7}, year = {2017}, month = {2017 Nov 15}, pages = {15600}, abstract = {

The conformational landscapes of p53 peptide variants\ and phage derived peptide (12/1) variants, all known to bind to MDM2, are studied using hamiltonian replica exchange molecular dynamics simulations. Complementing earlier observations, the current study suggests that the p53 peptides largely follow the {\textquoteright}conformational selection{\textquoteright} paradigm in their recognition of and complexation by MDM2 while the 12/1 peptides likely undergo some element of conformational selection but are mostly driven by {\textquoteright}binding induced folding{\textquoteright}. This hypothesis is further supported by pulling simulations that pull the peptides away from their bound states with MDM2. This data extends the earlier mechanisms proposed to rationalize the entropically driven binding of the p53 set and the enthalpically driven binding of the 12/1 set. Using our hypothesis, we suggest mutations to the 12/1 peptide that increase its helicity in simulations and may, in turn, shift the binding towards conformational selection. In summary, understanding the conformational landscapes of the MDM2-binding peptides may suggest new peptide designs with bespoke binding mechanisms.

}, issn = {2045-2322}, doi = {10.1038/s41598-017-15930-4}, author = {Yadahalli, Shilpa and Li, Jianguo and Lane, David P and Gosavi, Shachi and Verma, Chandra S} } @article {14979, title = {Packing energetics determine the folding routes of the RNase-H proteins.}, journal = {Phys Chem Chem Phys}, year = {2017}, month = {2017 Mar 20}, abstract = {

Comparative studies of proteins from a family have been used to understand the factors that determine the folding routes of proteins. It has been conjectured that the folding mechanism of ribonuclease-H (RNase-H) proteins is determined by the topology of their fold. To test this hypothesis, we computationally studied the folding of four proteins from the RNase-H family, which have the overall RNase-H fold, but whose topologies differ in the region termed CORE in E. coli RNase-H. We simulated the folding of these proteins using molecular dynamics (MD) simulations of a coarse-grained structure-based model (SBM) which captures the effects of topology and found that the four proteins had similar folding routes. However, these simulated folding routes do not agree with the folding routes of those RNase-H proteins that have been experimentally characterized. We next simulated the proteins using an SBM which specifically accounts for packing energetics and found that these routes not only vary substantially across the simulated RNase-H proteins but also agree with experiments. Thus, the packing energetics determine the folding mechanism of the RNase-H proteins. By comparing the differing folding routes calculated from the two models, we isolated packing interactions that promote these differences. We find that the balance of packing energetics between CORE and the rest of the protein is different across the different RNase-Hs. This balance determines the folding route. Our studies suggest that proteins from the RNase-H family should be used for experimentally detecting structurally distinct folding routes.

}, issn = {1463-9084}, doi = {10.1039/c6cp08940b}, author = {Yadahalli, Shilpa and Gosavi, Shachi} } @article {25264, title = {Understanding protein domain-swapping using structure-based models of protein folding.}, journal = {Prog Biophys Mol Biol}, volume = {128}, year = {2017}, month = {2017 Sep}, pages = {113-120}, abstract = {

In domain-swapping, two or more identical protein monomers exchange structural elements and fold into dimers or multimers whose units are structurally similar to the original monomer. Domain-swapping is of biotechnological interest because inhibiting domain-swapping can reduce disease-causing fibrillar protein aggregation. To achieve such inhibition, it is important to understand both the energetics that stabilize the domain-swapped structure and the protein dynamics that enable the swapping. Structure-based models (SBMs) encode the folded structure of the protein in their potential energy functions. SBMs have been successfully used to understand diverse aspects of monomer folding. Symmetrized SBMs model interactions between two identical protein chains using only intra-monomer interactions. Molecular dynamics simulations of such symmetrized SBMs have been used to correctly predict the domain-swapped structure and to understand the mechanism of domain-swapping. Here, we review such models and illustrate that monomer topology determines key aspects of domain-swapping. However, in some proteins, specifics of local energetic interactions modulate domain-swapping and these need to be added to the symmetrized SBMs. We then summarize some general principles of the mechanism of domain-swapping that emerge from the symmetrized SBM simulations. Finally, using our own results, we explore how symmetrized SBMs could be used to design domain-swapping in proteins.

}, keywords = {Models, Molecular, Protein Domains, Protein Folding, Proteins}, issn = {1873-1732}, doi = {10.1016/j.pbiomolbio.2016.09.013}, author = {Mascarenhas, Nahren Manuel and Gosavi, Shachi} } @article {11604, title = {Capturing the Membrane-Triggered Conformational Transition of an α-Helical Pore-Forming Toxin.}, journal = {J Phys Chem B}, volume = {120}, year = {2016}, month = {2016 Dec 01}, pages = {12064-12078}, abstract = {

Escherichia coli cytolysin A (ClyA) is an α-helical pore-forming toxin (PFT) which lyses target cells by forming membrane permeabilizing pores. The rate-determining step of this process is the conversion of the soluble ClyA monomer into a membrane inserted protomer. We elucidate the mechanism of this conformational transition using molecular dynamics simulations of coarse-grained models of ClyA and a membrane. We find that a membrane is necessary for the conformational conversion because membrane-protein interactions counteract the loss of the many intraprotein hydrophobic interactions that stabilize the membrane-inserting segments in the ClyA monomer. Of the two membrane-inserting segments, the flexible and highly hydrophobic β-tongue inserts first while the insertion of helix αA1 is membrane assisted. We conclude that the β-tongue is designed to behave as a quick-response membrane sensor, while helix αA1 improves target selectivity for cholesterol-containing cell membranes by acting as a fidelity check.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.6b09400}, author = {Giri Rao, V V Hemanth and Desikan, Rajat and Ayappa, K Ganapathy and Gosavi, Shachi} } @article {3553, title = {Functionally Relevant Specific Packing Can Determine Protein Folding Routes.}, journal = {J Mol Biol}, volume = {428}, year = {2016}, month = {2016 Jan 29}, pages = {509-21}, abstract = {

Functional residues can modulate the folding mechanisms of proteins. In some proteins, mutations to such residues can radically change the primary folding route. Is it possible then to learn more about the functional regions of a protein by investigating just its choice of folding route? The folding and the function of the protein Escherichia coli ribonuclease H (ecoRNase-H) have been extensively studied and its folding route is known to near-residue resolution. Here, we computationally study the folding of ecoRNase-H using molecular dynamics simulations of structure-based models of increasing complexity. The differences between a model that correctly predicts the experimentally determined folding route and a simpler model that does not can be attributed to a set of six aromatic residues clustered together in a region of the protein called CORE. This clustering, which we term "specific" packing, drives CORE to fold early and determines the folding route. Both the residues involved in specific packing and their packing are largely conserved across E. coli-like RNase-Hs from diverse species. Residue conservation is usually implicated in function. Here, the identified residues either are known to bind substrate in ecoRNase-H or pack against the substrate in the homologous human RNase-H where a substrate-bound crystal structure exists. Thus, the folding mechanism of ecoRNase-H is a byproduct of functional demands upon its sequence. Using our observations on specific packing, we suggest mutations to an engineered HIV RNase-H to make its function better. Our results show that understanding folding route choice in proteins can provide unexpected insights into their function.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2015.12.014}, author = {Yadahalli, Shilpa and Gosavi, Shachi} } @article {8357, title = {Protein Domain-Swapping Can Be a Consequence of Functional Residues.}, journal = {J Phys Chem B}, volume = {120}, year = {2016}, month = {2016 Jul 21}, pages = {6929-38}, abstract = {

Monomer topology has been implicated in domain-swapping, a potential first step on the route to disease-causing protein aggregation. Despite having the same topology (β1-α1-β2-β3-β4-β5), the cysteine protease inhibitor stefin-B domain swaps more readily than a single-chain variant of the heterodimeric sweet protein monellin (scMn). Here, we computationally study the folding of stefin-B and scMn in order to understand the molecular basis for the difference in their domain-swapping propensities. In agreement with experiments, our structure-based simulations show that scMn folds cooperatively without the population of an intermediate while stefin-B populates an equilibrium intermediate state. Since the simulation intermediate has only one domain structured (β3-β4-β5), it can directly lead to domain-swapping. Using computational variants of stefin-B, we show that the population of this intermediate is caused by regions of stefin-B that have been implicated in protease inhibition. We also find that the protease-binding regions are located on two structural elements and localized in space. In contrast, the residues that contribute to the sweetness of monellin are not localized to a few structural elements but are distributed over the protein fold. We conclude that the distributed functional residues of monellin do not induce large local perturbations in the protein structure, eliminating the formation of folding intermediates and in turn domain-swapping. On the other hand, the localized protease-binding regions of stefin-B promote the formation of a folding intermediate which can lead to domain-swapping. Thus, domain-swapping can be a direct consequence of the constraints that function imposes on the protein structure.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.6b03968}, author = {Mascarenhas, Nahren Manuel and Gosavi, Shachi} } @article {5310, title = {Using the folding landscapes of proteins to understand protein function.}, journal = {Curr Opin Struct Biol}, volume = {36}, year = {2016}, month = {2016 Feb}, pages = {67-74}, abstract = {

Proteins fold on a biologically-relevant timescale because of a funnel-shaped energy landscape. This landscape is sculpted through evolution by selecting amino-acid sequences that stabilize native interactions while suppressing stable non-native interactions that occur during folding. However, there is strong evolutionary selection for functional residues and these cannot be chosen to optimize folding. Their presence impacts the folding energy landscape in a variety of ways. Here, we survey the effects of functional residues on folding by providing several examples. We then review how such effects can be detected computationally and be used as assays for protein function. Overall, an understanding of how functional residues modulate folding should provide insights into the design of natural proteins and their homeostasis.

}, issn = {1879-033X}, doi = {10.1016/j.sbi.2016.01.001}, author = {Giri Rao, Vv Hemanth and Gosavi, Shachi} } @article {887, title = {Protein unfolding rates correlate as strongly as folding rates with native structure.}, journal = {Protein Sci}, volume = {24}, year = {2015}, month = {2015 Apr}, pages = {580-7}, abstract = {

Although the folding rates of proteins have been studied extensively, both experimentally and theoretically, and many native state topological parameters have been proposed to correlate with or predict these rates, unfolding rates have received much less attention. Moreover, unfolding rates have generally been thought either to not relate to native topology in the same manner as folding rates, perhaps depending on different topological parameters, or to be more difficult to predict. Using a dataset of 108 proteins including two-state and multistate folders, we find that both unfolding and folding rates correlate strongly, and comparably well, with well-established measures of native topology, the absolute contact order and the long range order, with correlation coefficient values of 0.75 or higher. In addition, compared to folding rates, the absolute values of unfolding rates vary more strongly with native topology, have a larger range of values, and correlate better with thermodynamic stability. Similar trends are observed for subsets of different protein structural classes. Taken together, these results suggest that choosing a scaffold for protein engineering may require a compromise between a simple topology that will fold sufficiently quickly but also unfold quickly, and a complex topology that will unfold slowly and hence have kinetic stability, but fold slowly. These observations, together with the established role of kinetic stability in determining resistance to thermal and chemical denaturation as well as proteases, have important implications for understanding fundamental aspects of protein unfolding and folding and for protein engineering and design.

}, issn = {1469-896X}, doi = {10.1002/pro.2606}, author = {Broom, Aron and Gosavi, Shachi and Meiering, Elizabeth M} } @article {1009, title = {Structural Perturbations Present in the Folding Cores of Interleukin-33 and Interleukin-1β Correlate to Differences in Their Function.}, journal = {J Phys Chem B}, volume = {119}, year = {2015}, month = {2015 Aug 27}, pages = {11203-14}, abstract = {

The interleukin-1 cytokines belong to the β-trefoil fold family and play a key role in immune responses to infections and injury. We simulate the structure-based models of two interleukin-1 cytokines, IL-33 and IL-1β, and find that IL-33 has a lower barrier to folding than IL-1β. We then design the folding motif (FM) of the β-trefoil fold and identify structural deviations of IL-33 and IL-1β from this FM. In previous work, we found that structural deviations from the FM that are large enough to lower folding free energy barriers were part of ligand binding sites. In contrast, we find that structural perturbations in IL-33 and IL-1β which reduce the folding free energy barrier are located in the folding core and do not bind ligands. In both proteins, such core residues are interleaved with surface residues which are proximal to receptor binding sites. However, IL-33 has a lower folding barrier because its core perturbations are larger than those in IL-1β. In order to understand the role of these core perturbations, we perform atomistic simulations of both proteins and find that the larger core perturbations may allow IL-33 to communicate signals differently across the protein. Integrating previous data, we also hypothesize that the larger IL-33 core perturbations may help accommodate its more charged binding site and may also aid in its inactivation by caspase-mediated cleavage. Together, our results suggest that protein folding landscapes are modulated not only by larger functional features such as binding sites but also by the details of protein function and fate. Furthermore, a comparative study of such landscapes may be a facile way to identify subtle differences in allosteric connectivity between two similar proteins.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.5b03111}, author = {Giri Rao, V V Hemanth and Gosavi, Shachi} }