Folding Mechanism of Beta-Hairpin Trpzip2: Heterogeneity, Transition State and Folding Pathways

We review the studies on the folding mechanism of the β-hairpin tryptophan zipper 2 (trpzip2) and present some additional computational results to refine the picture of folding heterogeneity and pathways. We show that trpzip2 can have a two-state or a multi-state folding pattern, depending on whether it folds within the native basin or through local state basins on the high-dimensional free energy surface; Trpzip2 can fold along different pathways according to the packing order of tryptophan pairs. We also point out some important problems related to the folding mechanism of trpzip2 that still need clarification, e.g., a wide distribution of the computed conformations for the transition state ensemble

Testing recently-developed molecular dynamics protocols for structure prediction of polypeptides and prion proteins.

Two MD protocols were recently developed that allow a polypeptide to search the PE surface in search of the global PE minimum, which should correspond to the experimental structure. alpha-helical secondary structures have previously been tested. We tested an additional alpha helix (C-peptide of ribonuclease A), and we extended the tests with two beta-hairpin secondary structures (tryptophan zipper 2 and the B1 domain(41--56) of protein G). For the C-peptide of ribonuclease A, the a helix was the dominate secondary structure, but a beta hairpin was found, which to our knowledge had not previously been reported. For the tryptophan zipper 2 and the B1 domain(41--56) of protein G, the beta hairpin was reproduced but alternative conformations were also found. After these test cases, we simulated a small protein (betabetaalpha5) that contained both secondary structural motifs and an overall tertiary structure. The secondary structures were reproduced, but the tertiary structure was not maintained. Finally, we attempted to predict possible conformations for a 64-residue protein, Ure2p, which is implicated in amyloid diseases of yeast. We found that Ure2p(1--64) was dominated by helical conformations. The DIVE and DIP protocols will need to be tested further with different polypeptides and proteins and using more recent force fields. Ure2p(1--64) should be simulated from additional secondary structures such as a beta sheet or a combination of alpha helices and beta hairpins

Using Molecular Constraints and Unnatural Amino Acids to Manipulate and Interrogate Protein Structure, Dynamics, and Self-Assembly

Protein molecules can undergo a wide variety of conformational transitions occurring over a series of time and distance scales, ranging from large-scale structural reorganizations required for folding to more localized and subtle motions required for function. Furthermore, the dynamics and mechanisms of such motions and transitions delicately depend on many factors and, as a result, it is not always easy, or even possible, to use existing experimental techniques to arrive at a molecular level understanding of the conformational event of interest. Therefore, this thesis aims to develop and utilize non-natural chemical modification strategies, namely molecular cross-linkers and unnatural amino acids as site-specific spectroscopic probes, in combination with various spectroscopic methods to examine, in great detail, certain aspects of protein folding and functional dynamics, and to manipulate protein self-assemblies. Specifically, we first demonstrate how strategically placed molecular constraints can be used to manipulate features of the protein folding free energy landscape, thus, allowing direct measurement of key components via temperature-jump kinetic studies, such as folding from a transition-state structure or the effect of internal friction on the folding mechanism. Secondly, we utilize a photolabile non-natural amino acid, Lys(nvoc), to probe the mechanism of protein misfolding in a β-hairpin model and identify an aggregation gatekeeper that tunes the aggregation propensity. We further develop a method where the induced-charge produced by photocleavage of Lys(nvoc) can be used to target and destabilize hydrophobic regions of amyloid fibril assemblies, resulting in complete disassembly, Finally, we highlight new useful properties of a site-specific spectroscopic probe, 5-cyanotryptophan (TrpCN), by demonstrating (1) how the frequency and linewidth of the infrared nitrile stretching vibration is sensitive to multiple hydrogen bonding interactions and solvent polarity, (2) that the fluorescence emission, quantum yield, and lifetime is extremely sensitive to hydration, and serves as a convenient fluorescence probe of protein solvation status, and (3) that the unique characteristics of TrpCN can be used to target the structure, local environment, and mechanism of the tryptophan gate in the M2 membrane proton channel of the influenza A virus

Markov state models of biomolecular conformational dynamics

It has recently become practical to construct Markov state models (MSMs) that reproduce the long-time statistical conformational dynamics of biomolecules using data from molecular dynamics simulations. MSMs can predict both stationary and kinetic quantities on long timescales (e.g. milliseconds) using a set of atomistic molecular dynamics simulations that are individually much shorter, thus addressing the well-known sampling problem in molecular dynamics simulation. In addition to providing predictive quantitative models, MSMs greatly facilitate both the extraction of insight into biomolecular mechanism (such as folding and functional dynamics) and quantitative comparison with single-molecule and ensemble kinetics experiments. A variety of methodological advances and software packages now bring the construction of these models closer to routine practice. Here, we review recent progress in this field, considering theoretical and methodological advances, new software tools, and recent applications of these approaches in several domains of biochemistry and biophysics, commenting on remaining challenges

Enhanced sampling and applications in protein folding

We show that a single-copy tempering method is useful in protein-folding simulations of large scale and high accuracy (explicit solvent, atomic representation, and physics-based potential). The method uses a runtime estimate of the average potential energy from an integral identity to guide a random walk in the continuous temperature space. It was used for folding three mini-proteins, trpzip2 (PDB ID: 1LE1), trp-cage (1L2Y), and villin headpiece (1VII) within atomic accuracy. Further, using a modification of the method with a dihedral bias potential added on the roof temperature, we were able to fold four larger helical proteins: α3D (2A3D), α3W (1LQ7), Fap1-NRα (2KUB) and S-836 (2JUA). We also discuss how to optimally use simulation data through an integral identity. With the help of a general mean force formula, the identity makes better use of data collected in a molecular dynamics simulation and is more accurate and precise than the common histogram approach

A global optimization approach for searching low energy conformations of proteins

De novo protein structure prediction and understanding the protein folding mechanism is an outstanding challenge of Biological Physics. Relying on the thermodynamic hypothesis of protein folding it is expected that the native state of a protein can be found out if the global minimum of the free energy surface is found. To understand the energy landscape or the free energy surface is challenging. The structure and dynamics of proteins are the manifestations of the underlying potential energy surface. Here the potential energy function stands on a framework of all-atom representation and uses purely physics-based interactions. For the solvated proteins the effective free energy is defined as an implicit solvation model which includes the solvation free energy, along with a standard all-atom biomolecular forcefield. A major challenge is to search for the global minimum on this effective free energy surface. In this work the Minima Hopping Algorithm (MHOP) to find global minima on potential energy surfaces has been used for protein structure prediction or in general finding the lowest energy conformations of proteins. Here proteins have been studied both in vacuo and in the aqueous medium. For short peptides starting from a completely extended conformation we could find conformational minima which are very close to the experimentally observed structures

Observing the unfolding transition of [beta]-hairpin peptides with nonlinear infrared spectroscopy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.In title on t.p., "[beta]" appears as the Greek letter. Vita.Includes bibliographical references.The biological function of a protein is in large measure determined by its three-dimensional structure. To date, however, the transition of the protein between the native and non-native conformations is not well-understood. Part of the difficulty is the large conformational space available to a poly-peptide chain, and a general lack of experimental probes that can access local structural information on the time scale of the transition. Single domain peptides are excellent model systems that reduce the size and complexity of the problem, while maintaining the essential physical interactions. In this thesis, P-hairpin peptides are used as model systems for studying P-sheet secondary structure. Hairpin folding has been studied for a number of years, but there is still debate in the literature about the relative importance of the cross-strand hydrogen bonds, tertiary side chain contacts, and p-turn in the folding pathway. In addition, the denatured state is very poorly understood, which complicates any attempt to describe the folding pathway. In this work, amide I vibrational spectroscopy is used to resolve the secondary structure of P-hairpin peptides during thermal denaturation. Spectroscopic modeling is presented to describe the amide I band of 0-hairpins and relate it to structural features. Three spectroscopic methods are used to probe the amide I band: Fourier transform infrared (FTIR) spectroscopy, two-dimensional infrared (2D IR) spectroscopy, and dispersed vibrational echo (DVE) spectroscopy. 2D IR and DVE spectroscopy are 3rd order-nonlinear methods that interrogate the system with a series of ultrafast (100 fs) laser pulses. 2D IR spectra reveal vibrational couplings and measure spectral dynamics on a picosecond time scale. .(cont.) The 2D IR spectra of TZ2 and PG12 are used to identify 3sheet structure during thermal denaturation and to measure the amide I homogeneous line width changes with temperature. The transient folding of TZ2 and PG12 is also probed with 2D IR and DVE spectroscopy following a 10 to 20 OC temperature jump. In order to increase the structural sensitivity of amide I spectroscopy, 13C and 180 isotope labels are incorporated into specific peptide amide groups. The isotope labels red-shift vibrational frequencies and help resolve local structure at the turn and mid-strand regions of the peptides. The transient folding at each labeled site is also measured following a temperature jump. Together, the results of this work identify folding rates for the thermal disordering transition. For PG12, the unfolding time at the mid-strand region of the peptide is 130 ns, and the turn is found to be stable throughout the transition. For TZ2, the kinetic folding rates at each of the labeled sites are found to be very similar to the global unfolding time (-1 Its). Temperature jump 2D IR spectroscopy of TZ2 reveals that the disordering mechanism is unique for different regions of the peptide. The band corresponding to the turn region decouples from the other vibrations, but does not show signs of disorder. In the mid-strand region of the peptide, the isotope-shifted band decouples from the main amide I band and also broadens significantly. Local disordering and decoupling both occur on a 1 gis time scale. The observations in this work combined with previous measurements are used to describe the folding as a hybrid zipper. TZ2 folding is initiated with the formation of the P-turn, following which the tryptophan side chains form a compact, but non-native hydrophobic core. Next, the backbone native contacts are formed and finally the tryptophan side chain packing reaches the native configuration.by Adam W. Smith.Ph.D

PyEMMA 2: A Software Package for Estimation, Validation, and Analysis of Markov Models

Markov (state) models (MSMs) and related models of molecular kinetics have recently received a surge of interest as they can systematically reconcile simulation data from either a few long or many short simulations and allow us to analyze the essential metastable structures, thermodynamics, and kinetics of the molecular system under investigation. However, the estimation, validation, and analysis of such models is far from trivial and involves sophisticated and often numerically sensitive methods. In this work we present the opensource Python package PyEMMA (http://pyemma.org) that provides accurate and efficient algorithms for kinetic model construction. PyEMMA can read all common molecular dynamics data formats, helps in the selection of input features, provides easy access to dimension reduction algorithms such as principal component analysis (PCA) and time-lagged independent component analysis (TICA) and clustering algorithms such as k-means, and contains estimators for MSMs, hidden Markov models, and several other models. Systematic model validation and error calculation methods are provided. PyEMMA offers a wealth of analysis functions such that the user can conveniently compute molecular observables of interest. We have derived a systematic and accurate way to coarse-grain MSMs to few states and to illustrate the structures of the metastable states of the system. Plotting functions to produce a manuscript-ready presentation of the results are available. In this work, we demonstrate the features of the software and show new methodological concepts and results produced by PyEMMA