Metastable Conformations Research Articles

Mapping the Excited‐State Potential Energy Surface of a Photomolecular Motor

AbstractA detailed understanding of the operation and efficiency of unidirectional photomolecular rotary motors is essential for their effective exploitation in molecular nanomachines. Unidirectional motion relies on light‐driven conversion from a stable (1 a) to a metastable (1 b) conformation, which then relaxes through a thermally driven helix inversion in the ground state. The excited‐state surface has thus far only been experimentally characterised for 1 a. Here we probe the metastable, 1 b, excited state, utilising ultrafast transient absorption and femtosecond stimulated Raman spectroscopy. These reveal that the “dark” excited‐state intermediate between 1 a and 1 b has a different lifetime and structure depending on the initial ground‐state conformation excited. This suggests that the reaction coordinate connecting 1 a to 1 b differs to that for the reverse photochemical process. The result is contrasted with earlier calculations.

Defining the Role of the L/I Zipper in the Stability of Paramyxo‐ and Pneumovirus F Proteins

Paramyxoviruses such as Hendra virus (HeV) and Measles virus (MeV) are important pathogens of worldwide significance. Others such as Parainfluenza virus (PIV5) are widely used as paramyxovirus model systems. Despite their prevalence, currently, no FDA‐approved vaccines or antiviral therapeutics are available against them. Paramyxoviruses consist of a single‐stranded negative sense RNA genome enveloped in a host‐derive membrane. Paramyxovirus glycoproteins play an important role in infectivity: the attachment protein (G, HN, or H) docks the viral particle to the host cell, and the fusion protein, (F), drives fusion with a host cell membrane, which allows the release of viral contents into the host cell. To promote successful fusion, the F protein undergoes dramatic, essentially irreversible conformational changes which must be carefully timed. To avoid unproductive triggering events, F is held in a metastable prefusion conformation until the right signal for fusion occurs. Previous studies in HeV F have shown that the transmembrane (TM) domain is important for prefusion stability with a leucine/isoleucine (L/I) zipper playing a key role. Furthermore, similar L/I zipper motifs are present across the paramyxovirus and the closely related pneumovirus families. To define the significance of the L/I zipper motifs in other paramyxo‐ and pneumovirus F proteins, we created a PIV5 F mutant protein which has alanine substitutions to the L/I zipper in the TM domain. Our preliminary data suggest that the mutant expresses; however, compared to wild type, which demonstrates a strong localization to the plasma membrane, the mutant shows a ubiquitous distribution in cells. This suggests that the mutant undergoes different trafficking events compared to the wild type. Further studies will be performed to quantify surface and total expression of the mutant, and also examine its stability and fusogenic activity. The effect of the L/I zipper will also be investigated in the context of Human metapneumovirus, a pneumovirus in order to determine whether the TM domain of paramyxo‐ and pneumoviruses present a broad therapeutic target.Support or Funding InformationWe gratefully acknowledge the NIH/NIAID for supporting our research grant R01 AI051517This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Narrower HOMO-LUMO gap attained by conformational switching through peripheral polyarylation in 1,4,5,8-tetraaza-9,10-anthraquinodimethanes

2,3,6,7,11,11,12,12-Octaphenyl-1,4,5,8-tetraaza-9,10-anthraquinodimethane was prepared as a sterically hindered molecule from 2,3,6,7-tetraphenyl-1,4,5,8-tetraaza-9,10-anthraquinone via bis(dibromomethylene) derivative through the quadruple Suzuki-Miyaura coupling reaction. An X-ray analysis revealed that steric repulsion is relieved mainly by twisting the exomethylene bonds, which is less common deformation for hindered quinodimethanes. According to the DFT calculations, the folded geometry with bent exomethylene bonds is the metastable conformer, and thus the nitrogen atoms at peri-positons and polyaryl substituents induce novel switching for the preferred geometry of 9,10-anthraquinodimethane skeleton from the common bent form to the twisted form, which causes a decrease in the HOMO-LUMO-gap energy by ca. 1 eV.

Biological Nanopores: Confined Spaces for Electrochemical Single-Molecule Analysis.

Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins. In this Account, we highlight recent developments of biological nanopores in DNA-based sensing and in studying the conformational structures of DNA and RNA. Furthermore, we introduce the application of biological nanopores to investigate the conformations of peptides affected by charge, length, and dipole moment and to study disease-related proteins' structures and aggregation transitions influenced by an inhibitor, a promoter, or an applied voltage. To improve the sensing ability of biological nanopores and further extend their application to a wider range of molecular sensing, we focus on exploring novel biological nanopores, such as aerolysin and Stable Protein 1. Aerolysin exhibits an especially high sensitivity for the detection of single oligonucleotides both in current separation and duration. Finally, to facilitate the use of nanopore measurements and statistical analysis, we develop an integrated current measurement system and an accurate data processing method for nanopore sensing. The unique geometric structure of a biological nanopore offers a distinct advantage as a nanosensor for single-molecule sensing. The construction of the pore entrance is responsible for capturing the target molecule, while the lumen region determines the translocation process of the single molecule. Since the capture of the target molecule is predominantly diffusion-limited, it is expected that the capture ability of the nanopore toward the target analyte could be effectively enhanced by site-directed mutations of key amino acids with desirable groups. Additionally, changing the side chains inside the wall of the biological nanopore could optimize the geometry of the pore and realize an optimal interaction between the single-molecule interface and the analyte. These improvements would allow for high spatial and current resolution of nanopore sensors, which would ensure the possibility of dynamic study of single biomolecules, including their metastable conformations, charge distributions, and interactions. In the future, data analysis with powerful algorithms will make it possible to automatically and statistically extract detailed information while an analyte translocates through the pore. We conclude that these improvements could have tremendous potential applications for nanopore sensing in the near future.

On the folding of a structurally complex protein to its metastable active state

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.

Analysis by metadynamics simulation of binding pathway of influenza virus M2 channel blockers.

M2 protein of influenza A virus is a proton channel spanning the viral envelope. Activity of this proton channel is required for uncoating of viral particles and equilibrating the pH across the trans Golgi apparatus, which prevents conformational change in hemagglutinin. Amantadine, an anti-influenza A virus drug, inhibits M2 proton channel activity by binding to the channel pore; however, most currently circulating influenza A viruses are amantadine-resistant. The most prevalent resistant mutation is a substitution from Ser31 to Asn31 in M2. Further atomistic analysis of ligand-M2 complexes is needed to provide new approaches for the design of novel M2 channel blockers. Here, the free energy profiles of the binding kinetics of M2 channel blockers were examined by well-tempered metadynamics simulations and it was found that amantadine first binds to Asp24 of S31 M2 and forms a metastable conformation. In contrast, the free energy profiles of adamantyl bromothiophene dual inhibitor with either S31 M2 or N31 M2 are broad funnel-shaped curves, suggesting that adamantyl bromothiophene does not form metastable complexes with M2. The trajectory of well-tempered metadynamics simulations revealed that steric hindrance between adamantyl bromothiophene and S31 M2 interrupts formation of a metastable conformation at Asp24 and that a halogen bond between the bromine atom and N31 is responsible for pulling down the ligand to the channel pore of N31 M2 in the absence of a metastable state. Binding pathways of M2 channel blockers to M2 are here proposed on the basis of these findings; they may provide new approaches to designing further M2 channel blockers.

How multi-scale structural biology elucidated context-dependent variability in ectodomain conformation along withthe ligand capture and release cycle for LDLR family members.

The low-density lipoprotein receptor (LDLR) and its homologs capture and internalize lipoproteins into the cell. Due to the fact that LDLR family members possess a modular ectodomain that undergoes dynamic conformational changes, multi-scale structural analysis has been performed so as to understand the ligand capture and release mechanism. For example, crystallographic analyses have provided models for boththe entire ectodomain and high-resolution structures of individual modules. In addition, nuclear magnetic resonance spectroscopic analyses have shown the rigidity and flexibility of inter-module linkers to restrict the mobility of ectodomain. Accumulated structural data suggest that the ectodomains of LDLR family members are flexible at the cell surface and switch between two metastable conformations, that is, the extended and contracted conformations. Recent structural analysis of ApoER2, a close homolog of LDLR, raised the possibility that the receptor binds with the ligand in the contracted conformation. After transport to an endosome by endocytosis, the receptor undergoes a conformational change to the closed conformation for completion of ligand release. In contrast, LDLR has been reported to adopt the extended conformation when it binds with a inhibitory regulator that recruits LDLR toward the degradation pathway. These findings support a mechanism of different ectodomain conformations for binding the ligand versus binding the regulatory protein. In this review, I provide an overview of studies that analyze the structural and biophysical properties of the ectodomains of LDLR family members and discuss a hypothetical model for ligand uptake and receptor recycling that integrates the known ectodomain conformational variability.

Probing the metastable state of influenza hemagglutinin

Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.

Calreticulin: Challenges Posed by the Intrinsically Disordered Nature of Calreticulin to the Study of Its Function.

Calreticulin is a Ca2+-binding chaperone protein, which resides mainly in the endoplasmic reticulum but also found in other cellular compartments including the plasma membrane. In addition to Ca2+, calreticulin binds and regulates almost all proteins and most of the mRNAs deciding their intracellular fate. The potential functions of calreticulin are so numerous that identification of all of them is becoming a nightmare. Still the recent discovery that patients affected by the Philadelphia-negative myeloproliferative disorders essential thrombocytemia or primary myelofibrosis not harboring JAK2 mutations carry instead calreticulin mutations disrupting its C-terminal domain has highlighted the clinical need to gain a deeper understanding of the biological activity of this protein. However, by contrast with other proteins, such as enzymes or transcription factors, the biological functions of which are strictly defined by a stable spatial structure imprinted by their amino acid sequence, calreticulin contains intrinsically disordered regions, the structure of which represents a highly dynamic conformational ensemble characterized by constant changes between several metastable conformations in response to a variety of environmental cues. This article will illustrate the Theory of calreticulin as an intrinsically disordered protein and discuss the Hypothesis that the dynamic conformational changes to which calreticulin may be subjected by environmental cues, by promoting or restricting the exposure of its active sites, may affect its function under normal and pathological conditions.

Structure and immunogenicity of pre-fusion-stabilized human metapneumovirus F glycoprotein

Human metapneumovirus (hMPV) is a frequent cause of bronchiolitis in young children. Its F glycoprotein mediates virus–cell membrane fusion and is the primary target of neutralizing antibodies. The inability to produce recombinant hMPV F glycoprotein in the metastable pre-fusion conformation has hindered structural and immunological studies. Here, we engineer a pre-fusion-stabilized hMPV F ectodomain and determine its crystal structure to 2.6 Å resolution. This structure reveals molecular determinants of strain-dependent acid-induced fusion, as well as insights into refolding from pre- to post-fusion conformations. A dense glycan shield at the apex of pre-fusion hMPV F suggests that antibodies against this site may not be elicited by host immune responses, which is confirmed by depletion studies of human immunoglobulins and by mouse immunizations. This is a major difference with pre-fusion F from human respiratory syncytial virus (hRSV), and collectively our results should facilitate development of effective hMPV vaccine candidates.

Variational Identification of Markovian Transition States

We present a new method that enables the identification and analysis of both transition and metastable conformational states from atomistic or coarse-grained molecular dynamics (MD) trajectories. Our algorithm is presented and studied by using both analytical and actual examples from MD simulations of the helix-forming peptide Ala5, and of a larger system, the epidermal growth factor receptor (EGFR) protein. In all cases, our method identifies automatically the corresponding transition states and metastable conformations in an optimal way, with the input of a set of relevant coordinates, by capturing accurately the intrinsic slowest relaxation rate. Our approach provides a general and easy to implement analysis method that provides unique insight into the molecular mechanism and the rare but crucial rate limiting conformational pathways occurring in complex dynamical systems such as molecular trajectories.

Single-molecule analysis in an electrochemical confined space

Electrochemical analysis of single molecules is a method with the strong ability of the enhanced efficiency and ultra-sensitivity. Here, we demonstrate that the electrochemical confined space could efficiently convert single molecule characteristics into measurable electrochemical signatures with high temporal resolution. The human telomere repeat sequence T8 was used as a probe to determine the electrochemical confined effect in a nanopore. Our results show that the nanopore with comparable confined space of the telomere repeat sequence exhibits the most distinguishable single-molecule signals which suggest the folded conformation of T8. This method will greatly extend the lifetime of a metastable conformation for a single biomolecule by strong analyte-nanopore interactions, which brings the new insight into the understanding of the biomolecule’s function at single-molecule level.

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.

Effectively explore metastable states of proteins by adaptive nonequilibrium driving simulations

Nonequilibrium drivings applied in molecular dynamics (MD) simulations can efficiently extend the visiting range of protein conformations, but might compel systems to go far away from equilibrium and thus mainly explore irrelevant conformations. Here we propose a general method, called adaptive nonequilibrium simulation (ANES), to automatically adjust the external driving on the fly, based on the feedback of the short-time average response of system. Thus, the ANES approximately keeps the local equilibrium but efficiently accelerates the global motion. We illustrate the capability of the ANES in highly efficiently exploring metastable conformations in the deca-alanine peptide and find that the 0.2-μs ANES approximately captures the important states and folding and unfolding pathways in the HP35 solution by comparing with the result of the recent 398-μs equilibrium MD simulation on Anton [S. Piana et al., Proc. Natl. Acad. Sci. USA 109, 17845 (2012)PNASA60027-842410.1073/pnas.1201811109].

Metastability Phenomena in VO2 Thin Films

VO2 is a transition metal oxide in which complex electronic phases appear near the metal-to-insulator transition due to electron correlation and electron–lattice interactions. This system is characterized by a metal-to-insulator transition (MIT) at around 341 K. The metal (high T) phase is tetragonal while the insulator (low T) phase is monoclinic and the resistivity changes at the MIT by about five orders of magnitude. Here, we report investigations of the MIT in a thin VO2 film deposited on a sapphire substrate showing hysteresis. The MIT has been characterized by resistance measurements versus temperature and a DC magnetic field. The thin sample shows different final resistance values in both the insulating and metallic state after different temperature cycles. Moreover, some cycles do not close in the insulating phase. An unexpected magnetic dependence of the temperature cycle in the sample was also observed. The results show that the MIT of VO2 can be controlled by reducing the thickness below 40 nm in micron-sized ribbons since MIT is associated with the emergence of coexisting metastable conformations controlled by the thickness-dependent misfit strain and stress distributions induced by the mismatch between thin ribbon film and the substrate.

NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations.

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

Finding Protein Folding Funnels in Random Networks

Many attempts have been made for understanding protein folding dynamics using network representations of folding pathway. For example, the Markov state model has been widely used in analyzing the transitions between protein conformations of trajectories obtained by molecular dynamics simulations.In the present work, I also focus on the network structures of protein folding dynamics, but from a somewhat different perspective. Currently most widely accepted theoretical framework of the protein folding is so-called funnel picture, which states that the number of conformations decreases as the energy lowers from the denatured state towards the native state. In other words, the energy landscapes of proteins have been designed through Darwinian evolution so that proteins readily fold to their native states. A question I would like to ask here is “How rare are such folding-friendly funnel structures?”.As a first attempt to answer this question, I introduce a random network model with each nodes assigned a random energy. Nodes represent the metastable conformation ensembles. One of the nodes is considered as denatured state and another the folded state. Other nodes represent the intermediate conformation ensembles between them. Edge connecting the nodes are possible transitions between the nodes. The network structure is assumed to be determined by the native state conformation, and the energy assignment is considered to reflects the amino-acid sequence. We then define the ideal funnel structure for this model as follows: Starting from the denatured state, if all the paths connecting the nodes in energy-lowering direction reach the folded state, then such a network is in a ideal funnel structure. The task now is to compute the probability that the ideal funnel is realized for a given network by changing the assignment of energy.We generated 1000 random networks for number of nodes from 8 to 16. The probability is widely distributed from network to network, and close to the log-normal distribution . This fact means that there are a small number of networks that are robust against the mutation. The funnel structure becomes rarer exponentially as the protein becomes longer.

Excited state dynamics and conformations of a Cu(ii)-phthalocyanine-perylenebisimide dyad.

We investigate the excited state dynamics and the conformations of a new molecular donor-bridge-acceptor system, a Cu(ii)-phthalocyanine (CuPc) covalently linked via a flexible aliphatic spacer to a perylenebisimide (PBI). We performed time-resolved polarization anisotropy and pump-probe measurements in combination with molecular dynamics simulations. Our data suggest the existence of three conformations of the dyad: two more extended, metastable conformations with centre-of-mass distances >1 nm between the PBI and CuPc units of the dyad, and a highly stable folded structure, in which the PBI and CuPc units are stacked on top of each other with a centre-of-mass distance of 0.4 nm. In the extended conformations the dyad shows emission predominantly from the PBI unit with a very weak contribution from the CuPc unit. In contrast, for the folded conformation the PBI emission of the dyad is strongly quenched due to fast energy transfer from the PBI to the CuPc unit (3 ps) and subsequent intersystem-crossing (300 fs) from the first excited singlet state of CuPc unit into its triplet state. Finally, the CuPc triplet state is deactivated non-radiatively with a time constant of 25 ns.

Polymorphism at 129 dictates metastable conformations of the human prion protein N-terminal β-sheet.

We study the thermodynamic stability of the native state of the human prion protein using a new free-energy method, replica-exchange on-the-fly parameterization. This method is designed to overcome hidden-variable sampling limitations to yield nearly error-free free-energy profiles along a conformational coordinate. We confirm that all four (M129V, D178N) polymorphs have a ground-state conformation with three intact β-sheet hydrogen bonds. Additionally, they are observed to have distinct metastabilities determined by the side-chain at position 129. We rationalize these findings with reference to the prion "strain" hypothesis, which links the variety of transmissible spongiform encephalopathy phenotypes to conformationally distinct infectious prion forms and classifies distinct phenotypes of sporadic Creutzfeldt-Jakob disease based solely on the 129 polymorphism. Because such metastable structures are not easily observed in structural experiments, our approach could potentially provide new insights into the conformational origins of prion diseases and other pathologies arising from protein misfolding and aggregation.

Finite Temperature String Method with Umbrella Sampling: Application on a Side Chain Flipping in Mhp1 Transporter.

Protein conformational change is of central importance in molecular biology. Here we demonstrate a computational approach to characterize the transition between two metastable conformations in all-atom simulations. Our approach is based on the finite temperature string method, and the implementation is essentially a generalization of umbrella sampling simulations with Hamiltonian replica exchange. We represent the transition pathway by a curve in the conformational space, with the curve parameter taken as the reaction coordinate. Our approach can efficiently refine a transition pathway and compute a one-dimensional free energy as a function of the reaction coordinate. A diffusion model can then be used to calculate the forward and backward transition rates, the major kinetic quantities for the transition. We applied the approach on a local transition in the ligand-free Mhp1 transporter, between its outward-facing conformation and an intermediate conformation with the side chain of Phe305 flipped to the outside of the protein. Our simulations predict that the flipped-out position of this side chain has a free energy 6.5 kcal/mol higher than the original position in the crystal structure, and that the forward and backward transition rates are in the millisecond and submicrosecond time scales, respectively.