Observation of Fragile-to-Strong Dynamic Crossover in Protein Hydration Water

At low temperatures proteins exist in a glassy state, a state which has no conformational flexibility and shows no biological functions. In a hydrated protein, at and above 220 K, this flexibility is restored and the protein is able to sample more conformational sub-states, thus becomes biologically functional. This 'dynamical' transition of protein is believed to be triggered by its strong coupling with the hydration water, which also shows a similar dynamic transition. Here we demonstrate experimentally that this sudden switch in dynamic behavior of the hydration water on lysozyme occurs precisely at 220 K and can be described as a Fragile-to-Strong dynamic crossover (FSC). At FSC, the structure of hydration water makes a transition from predominantly high-density (more fluid state) to low-density (less fluid state) forms derived from existence of the second critical point at an elevated pressure.Comment: 6 pages (Latex), 4 figures (Postscript

Deciphering the 'fuzzy' interaction of FG nucleoporins and transport factors using SANS

The largely intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG Nups) underline a selectivity mechanism, which enables the rapid translocation of transport factors (TFs) through the nuclear pore complexes (NPCs). Conflicting models of NPC transport have assumed that FG Nups undergo different conformational transitions upon interacting with TFs. To selectively characterize conformational changes in FG Nups induced by TFs we performed small-angle neutron scattering (SANS) with contrast matching. Conformational ensembles derived SANS data indicate an increase in the overall size of FG Nups is associated with TF interaction. Moreover, the organization of the FG motif in the interacting state is consistent with prior experimental analyses defining that FG motifs undergo conformational restriction upon interacting with TFs. These results provide structural insights into a highly dynamic interaction and illustrate how functional disorder imparts rapid and selective FG Nup / TF interactions.Comment: Minor revisions and reformattin

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle

Detection of Side Chain Rearrangements Mediating the Motions of Transmembrane Helices in Molecular Dynamics Simulations of G Protein-Coupled Receptors.

Structure and dynamics are essential elements of protein function. Protein structure is constantly fluctuating and undergoing conformational changes, which are captured by molecular dynamics (MD) simulations. We introduce a computational framework that provides a compact representation of the dynamic conformational space of biomolecular simulations. This method presents a systematic approach designed to reduce the large MD simulation spatiotemporal datasets into a manageable set in order to guide our understanding of how protein mechanics emerge from side chain organization and dynamic reorganization. We focus on the detection of side chain interactions that undergo rearrangements mediating global domain motions and vice versa. Side chain rearrangements are extracted from side chain interactions that undergo well-defined abrupt and persistent changes in distance time series using Gaussian mixture models, whereas global domain motions are detected using dynamic cross-correlation. Both side chain rearrangements and global domain motions represent the dynamic components of the protein MD simulation, and are both mapped into a network where they are connected based on their degree of coupling. This method allows for the study of allosteric communication in proteins by mapping out the protein dynamics into an intramolecular network to reduce the large simulation data into a manageable set of communities composed of coupled side chain rearrangements and global domain motions. This computational framework is suitable for the study of tightly packed proteins, such as G protein-coupled receptors, and we present an application on a seven microseconds MD trajectory of CC chemokine receptor 7 (CCR7) bound to its ligand CCL21

Nonparametric inference of doubly stochastic Poisson process data via the kernel method

Doubly stochastic Poisson processes, also known as the Cox processes, frequently occur in various scientific fields. In this article, motivated primarily by analyzing Cox process data in biophysics, we propose a nonparametric kernel-based inference method. We conduct a detailed study, including an asymptotic analysis, of the proposed method, and provide guidelines for its practical use, introducing a fast and stable regression method for bandwidth selection. We apply our method to real photon arrival data from recent single-molecule biophysical experiments, investigating proteins' conformational dynamics. Our result shows that conformational fluctuation is widely present in protein systems, and that the fluctuation covers a broad range of time scales, highlighting the dynamic and complex nature of proteins' structure.Comment: Published in at http://dx.doi.org/10.1214/10-AOAS352 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

Dynamic phase transition in the conversion of B-DNA to Z-DNA

The long time dynamics of the conformational transition from B-DNA to Z-DNA is shown to undergo a dynamic phase transition. We obtained the dynamic phase diagram for the stability of the front separating B and Z. The instability in this front results in two split fronts moving with different velocities. Hence, depending on the system parameters a denatured state may develop dynamically eventhough it is thermodynamically forbidden. This resolves the current controversies on the transition mechanism of the B-DNA to Z-DNA.Comment: 5 pages, 4 figures. New version with correction of typos, new references, minor modifications in Fig 2, 3. To appear in EP

Gating mechanism of elongating β-ketoacyl-ACP synthases.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs