Conformational Trajectory Research Articles

Crystal Structures of the MthK RCK Domain Reveal Allosteric Interactions Among Calcium Binding Sites

Regulator of K+ conductance (RCK) domains form a conserved class of modulatory domains that undergo conformational changes with binding of metal cations and other ligands, to control gating of channels and transporters. In MthK, a prototypical RCK-containing K+ channel, each of the channel's eight RCK domains binds multiple Ca2+ ions to reach the fully-activated state, which can give rise to a complex conformational trajectory. Here we present crystal structures of the MthK RCK domain bound with Ca2+ in a series of singly-, doubly-, and triply-liganded states. These structures begin to reveal local conformational changes in this RCK domain that may arise from binding of Ca2+ at individual sites and pairs of sites over a range of ionic conditions, providing insight toward interactions among the sites that may modulate channel gating. Crystals formed at low to moderate [Ca2+] show Ca2+ bound only at a single site, termed C1, determined by residues D184, E210, and E212. In contrast, high [Ca2+] (in otherwise identical conditions) results in a new crystal form, with Ca2+ bound at sites C1, C2 (near residues E248 and E266), and C3 (residues D305 and E326). The mutation D184N, which abolishes Ca2+ binding at C1, permits Ca2+ binding at C3 with moderate [Ca2+], suggesting that Ca2+ binding at C1 inhibits binding at C3. This apparent negative coupling between sites C1 and C3 can be alleviated by the mutation E212Q, which permits Ca2+ binding at both C1 and C3 and facilitates Ca2+-dependent activation. These results suggest a structural basis for allosteric interactions that, in turn, modulate Ca2+℧dependent gating of the MthK channel.

Hydrogen Exchange Mass Spectrometry of Bacteriorhodopsin Reveals Light-Induced Changes in the Structural Dynamics of a Biomolecular Machine

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions.

Time-Resolved WAXS Reveals Accelerated Conformational Changes in Iodoretinal-Substituted Proteorhodopsin

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.

Real-time multidimensional NMR follows RNA folding with second resolution

Conformational transitions and structural rearrangements are central to the function of many RNAs yet remain poorly understood. We have used ultrafast multidimensional NMR techniques to monitor the adenine-induced folding of an adenine-sensing riboswitch in real time, with nucleotide-resolved resolution. By following changes in 2D spectra at rates of approximately 0.5 Hz, we identify distinct steps associated with the ligand-induced folding of the riboswitch. Following recognition of the ligand, long range loop-loop interactions form and are then progressively stabilized before the formation of a fully stable complex over approximately 2-3 minutes. The application of these ultrafast multidimensional NMR methods provides the opportunity to determine the structure of RNA folding intermediates and conformational trajectories.

Computational Search of Evidence for the Division of Amino Acids into Transitional Groups in a Virtual State Protein. Phase I: Equilibrium Fluctuations

Core-Shell model postulates that beyond certain resolution, non-equilibrium states are virtual, non-testable as proteinous, because they are not “domesticated” by evolution. We propose that during (un)folding, the {Cα, Cβ, Cγ, Cδ, Cɛ}-backbone of residue does an elementary move, transverse to peptide backbone. Sliding mechanism assigns amino acids into three transition groups based on residue stereochemistry. According to model, this division is masked in folded conformation. Search of evidence for such division begins with study of fluctuation of folded molecule, using united-residue protein model of sperm whale myoglobin to simulate Monte Carlo conformational trajectories. For alpha-carbon fluctuations along polypeptide chain, reasonable qualitative agreement between simulated and crystallographic B-factor (PDB ID: 108M) profiles is reached. Nearly equal amount of average rms-fluctuation contribution is found for T-groups: T1 (1.13±0.12A); T2 (1.17±0.14A); T3 (1.18±0.09A); with 1.13±0.11A being for the whole molecule. Model predicts constant-rate built-up of rms-fluctuation amounts as protein unfolds. Myoglobin has high symmetry of architecture and unusual sextet of amino acid pairs. For comparison, their rms-fluctuation amounts are: [Trp(2),Asn(2)] (1.38±0.57A); [Met(3),Tyr(3)] (0.85±0.00A); [Pro(4),Arg(4)] (1.25±0.14A); [Gln(5),Thr(5)] (1.14±0.09A); [Ser(6),Asp(6)] (1.25±0.18A); [Phe(7),Val(7)] (1.08±0.12A).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Procrustean rotation in concert with principal component analysis of molecular dynamics trajectories: Quantifying global and local differences between conformational samples

Given the principal component analysis (PCA) of a molecular dynamics (MD) conformational trajectory for a model protein, we perform orthogonal Procrustean rotation to "best fit" the PCA squared-loading matrix to that of a target matrix computed for a related but different molecular system. The sum of squared deviations of the elements of the rotated matrix from those of the target, known as the error of fit (EOF), provides a quantitative measure of the dissimilarity between the two conformational samples. To estimate precision of the EOF, we perform bootstrap resampling of the molecular conformations within the trajectories, generating a distribution of EOF values for the system and target. The average EOF per variable is determined and visualized to ascertain where, locally, system and target sample properties differ. We illustrate this approach by analyzing MD trajectories for the wild-type and four selected mutants of the beta1 domain of protein G.

Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses

Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 Å resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes.

Residue Level Three-dimensional Workspace Maps for Conformational Trajectory Planning of Proteins

The function of a protein macromolecule often requires conformational transitions between two native conformations. Understanding these transitions is essential to the understanding of how proteins function, as well as to the ability to design and manipulate protein-based nanomechanical systems. In this paper we propose a set of 3D Cartesian workspace maps for exploring protein pathways. These 3D maps are constructed in the Euclidean space for triads of chain segments of protein molecules that have been shown to have a high probability of occurrence in naturally observed proteins based on data obtained from more than 38,600 proteins from the Protein Data Bank (PDB). We show that the proposed 3D propensity maps are more effective navigation tools than the propensity maps constructed in the angle space. We argue that the main reason for this improved efficiency is the fact that, although there is a one-to-one mapping between the 2D dihedral angle maps and the 3D Cartesian maps, the propensity distributions are significantly different in the two spaces. Hence, the 3D maps allow the pathway planning to be performed directly in the 3D Euclidean space based on propensities computed in the same space, which is where protein molecules change their conformations.

Extent of Voltage Sensor Movement during Gating of Shaker K + Channels

Voltage-driven activation of Kv channels results from conformational changes of four voltage sensor domains (VSDs) that surround the K(+) selective pore domain. How the VSD helices rearrange during gating is an area of active research. Luminescence resonance energy transfer (LRET) is a powerful spectroscopic ruler uniquely suitable for addressing the conformational trajectory of these helices. Using a geometric analysis of numerous LRET measurements, we were able to estimate LRET probe positions relative to existing structural models. The experimental movement of helix S4 does not support a large 15-20 A transmembrane "paddle-type" movement or a near-zero A vertical "transporter-type" model. Rather, our measurements demonstrate a moderate S4 displacement of 10 +/- 5 A, with a vertical component of 5 +/- 2 A. The S3 segment moves 2 +/- 1 A in the opposite direction and is therefore not moving as an S3-S4 rigid body.

Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed inPichia pastoris

The amphiphilic saposin proteins (A, B, C and D) act at the lipid-water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and beta-galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 A. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins.

ChemInform Abstract: The Effect of Harmonic Conformational Trajectories on Protein Fluorescence and Lifetime Distributions.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The effect of harmonic conformational trajectories on protein fluorescence and lifetime distributions

Proteins change their structure constantly due to their conformational flexibility and dynamic nature. The structure of the molecule follows a trajectory in conformational space determined by the hierarchy of conformational substates of the protein. These conformational trajectories may differ in the ground and excited states. The molecule constant structural changes exposes the fluorescent protein residues to continuously mutating environments in a predetermined sequence given by the molecule conformational path. The absorption extinction coefficient and the emission decay rate of a given fluorescent residue, dependent upon the instantaneous localized environment of the residue, are modulated by the conformational dynamics of the molecule. A simple harmonic conformational trajectory describes the behavior of traditional, time invariant, fluorescence lifetime distributions with temperature. The lifetime distribution describes at any given time the distribution of excited residues in the protein conformational space. Consequently, dynamic conformational trajectories require the use of time dependent lifetime distributions. The evolution of uniform lifetime distributions modulated by harmonic conformational trajectories are illustrated. The results presented here open the way to the description of complex conformational dynamics in terms of structural harmonic fluctuations.