Kevin Y. Sanbonmatsu

α-Helical stabilization by side chain shielding of backbone hydrogen bonds

We study atomic models of the thermodynamics of the structural transition of peptides that form α-helices. The effect of sequence variation on α-helix formation for alanine-rich peptides, Ac-Ala21- methyl amide (A21) and Ac-A5 (AAARA)3A-methyl amide (Fs peptide), is investigated by atomic simulation studies of the thermodynamics of the helix-coil transition in explicit water. The simulations show that the guanidinium group in the Arg side chains in the Fs peptide interacts with the carbonyl group four amino acids upstream in the chain and desolvates backbone hydrogen bonds. This desolvation can be directly correlated with a higher probability of hydrogen bond formation. We find that Fs has higher helical content than A21 at all temperatures. A small modification in the amber force field reproduces the experimental helical content and helix-coil transition temperatures for the Fs peptide.

Exploring the energy landscape of a β hairpin in explicit solvent

We studied the energy landscape of the peptide Ace‐GEWTYDDATKTFTVTE‐Nme, taken from the C‐terminal fragment (41–56) of protein G, in explicit aqueous solution by a highly parallel replica‐exchange approach that combines molecular dynamics trajectories with a temperature exchange Monte Carlo process. The combined trajectories in T and configurational space allow a replica to overcome a free energy barrier present at one temperature by increasing T, changing configurations, and cooling in a self‐regulated manner, thus allowing sampling of broad regions of configurational space in short (nanoseconds) time scales. The free energy landscape of this system over a wide range of temperatures shows that the system preferentially adopts a beta hairpin structure. However, the peptide also samples other stable ensembles where the peptide adopts helices and helix‐turn‐helix states, among others. The helical states become increasingly stable at low temperatures, but are slightly less stable than the beta turn ensemble. The energy landscape is rugged at low T, where substates are separated by large energy barriers. These barriers disappear at higher T (∼330 K), where the system preferentially adopts a “molten globule” state with structures similar to the beta hairpin. Proteins 2001;42:345–354.

An all‐atom structure‐based potential for proteins: Bridging minimal models with all‐atom empirical forcefields

Protein dynamics take place on many time and length scales. Coarse‐grained structure‐based

Structure of Met-enkephalin in explicit aqueous solution using replica exchange molecular dynamics.

{\bf (G{\overline o})}

Peptide folding simulations

models utilize the funneled energy landscape theory of protein folding to provide an understanding of both long time and long length scale dynamics. All‐atom empirical forcefields with explicit solvent can elucidate our understanding of short time dynamics with high energetic and structural resolution. Thus, structure‐based models with atomic details included can be used to bridge our understanding between these two approaches. We report on the robustness of folding mechanisms in one such all‐atom model. Results for the B domain of Protein A, the SH3 domain of C‐Src Kinase, and Chymotrypsin Inhibitor 2 are reported. The interplay between side chain packing and backbone folding is explored. We also compare this model to a Cα structure‐based model and an all‐atom empirical forcefield. Key findings include: (1) backbone collapse is accompanied by partial side chain packing in a cooperative transition and residual side chain packing occurs gradually with decreasing temperature, (2) folding mechanisms are robust to variations of the energetic parameters, (3) protein folding free‐energy barriers can be manipulated through parametric modifications, (4) the global folding mechanisms in a Cα model and the all‐atom model agree, although differences can be attributed to energetic heterogeneity in the all‐atom model, and (5) proline residues have significant effects on folding mechanisms, independent of isomerization effects. Because this structure‐based model has atomic resolution, this work lays the foundation for future studies to probe the contributions of specific energetic factors on protein folding and function. Proteins 2009.

Spontaneous formation of the unlocked state of the ribosome is a multistep process

Replica exchange molecular dynamics (MD) simulations of Met‐enkephalin in explicit solvent reveal helical and nonhelical structures. Four predominant structures of Met‐enkephalin are sampled with comparable probabilities (two helical and two nonhelical). The energy barriers between these configurations are low, suggesting that Met‐enkephalin switches easily between configurations. This is consistent with the requirement that Met‐enkephalin be sufficiently flexible to bind to several different receptors. Replica exchange simulations of 32 ns are shown to sample approximately five times more configurational space than constant temperature MD simulations of the same duration. The energy landscape for the replica exchange simulation is presented. A detailed study of replica trajectories demonstrates that the significant increases in temperature provided by the replica exchange technique enable transitions from nonhelical to helical structures that would otherwise be prevented by kinetic trapping. Met‐enkephalin (Type Entrez Proteins; Value A61445; Service Entrez Proteins) Proteins 2002;46:225–234. Published 2001 Wiley‐Liss, Inc.

A fast dynamic mode of the EF-G-bound ribosome

Developments in the design of small peptides that mimic proteins in complexity, recent advances in nanosecond time-resolved spectroscopy methods to study peptides and the development of modern, highly parallel simulation algorithms have come together to give us a detailed picture of peptide folding dynamics. Two newly implemented simulation techniques, parallel replica dynamics and replica exchange molecular dynamics, can now describe directly from simulations the kinetics and thermodynamics of peptide formation, respectively. Given these developments, the simulation community now has the tools to verify and validate simulation protocols and models (forcefields).

Understanding Discrimination by the Ribosome: Stability Testing and Groove Measurement of Codon- Anticodon Pairs

The mechanism of substrate translocation through the ribosome is central to the rapid and faithful translation of mRNA into proteins. The rate-limiting step in translocation is an unlocking process that includes the formation of an “unlocked” intermediate state, which requires the convergence of large-scale conformational events within the ribosome including tRNA hybrid states formation, closure of the ribosomal L1 stalk domain, and subunit ratcheting. Here, by imaging of the pretranslocation ribosome complex from multiple structural perspectives using two- and three-color single-molecule fluorescence resonance energy transfer, we observe that tRNA hybrid states formation and L1 stalk closure, events central to the unlocking mechanism, are not tightly coupled. These findings reveal that the unlocked state is achieved through a stochastic-multistep process, where the extent of conformational coupling depends on the nature of tRNA substrates. These data suggest that cellular mechanisms affecting the coupling of conformational processes on the ribosome may regulate the process of translation elongation.

All-atom homology model of the Escherichia coli 30S ribosomal subunit

A key intermediate in translocation is an ‘unlocked state’ of the pre‐translocation ribosome in which the P‐site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two‐ and three‐colour smFRET imaging from multiple structural perspectives, EF‐G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF‐G‐bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.

Stochastic gating and drug-ribosome interactions.

The ribosome must discriminate between correct and incorrect tRNAs with sufficient speed and accuracy to sustain an adequate rate of cell growth. Here, we report the results of explicit solvent molecular dynamics simulations, which address the mechanism of discrimination by the ribosome. The universally conserved 16S rRNA base A1493 and the kink in mRNA between A and P sites amplify differences in stability between cognate and near-cognate codon-anticodon pairs. Destabilization by the mRNA kink also provides a geometric explanation for the higher error rates observed for mismatches in the first codon position relative to mismatches in the second codon position. For more stable near-cognates, the repositioning of the universally conserved bases A1492 and G530 results in increased solvent exposure and an uncompensated loss of hydrogen bonds, preventing correct codon-anticodon-ribosome interactions from forming.