We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air-water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer-water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins.osmolytes | protein folding | mechanism | spectroscopy | MD simulations M any organisms use small organic osmolytes to stabilize proteins in harsh environments, such as when the salinity is highly variable (1). In particular, trimethylamine N-oxide (TMAO) is known to counteract the denaturing effects of urea as well as salts, and it is present at high concentrations in some aquatic organisms (2). Its effects are often compared with the ions on the left side of the Hofmeister series, which help stabilize the native, folded structures of proteins (Fig. 1).Because of their fundamental biophysical importance, many studies have investigated the behavior and effects of osmolytes. In particular, Timasheff and coworkers (3, 4) proposed that osmolyte effects result from the relative partitioning of these molecules between the bulk solution and the protein-water interface. Stabilization should occur when osmolytes are depleted from the protein-water interface, but proteins will unfold when osmolytes accumulate at this interface. Accordingly, osmolyte effects are often interpreted in terms of an effective protein-water "surface tension." In fact, despite the significant differences between protein surfaces and air-water interfaces, osmolyte effects are often, although not always, consistent with their effect on the air-water interfa...

Observations of front geometry and refractive index jump across shock waves in rare gases have been made with a new and particularly simple technique. The technique involves determination of the angular deflection of a narrow laser beam intersecting the shock front at a shallow angle. Measured refractive index jumps in rare gases are in excellent agreement with those calculated using Snell’s law and ideal shock theory. The apparent shock curvature is in close accord with deBoer’s theory for loading pressures below 20 Torr, but above this presure there is evidence of an indentation near tube center.

Protein conformational fluctuations are key contributors to biological function, mediating important processes such as enzyme catalysis, molecular recognition and allosteric signaling. To better understand the role of conformational fluctuations in substrate:ligand recognition, we analyzed, experimentally and computationally, the binding reaction between an SH3 domain and the recognition peptide of its partner protein. The fluctuations in this SH3 domain were enumerated by using an algorithm based on the hard sphere collision model, and the binding energetics resulting from these fluctuations were calculated using a structure-based energy function parameterized to solvent accessible surface areas. Surprisingly, this simple model reproduced the effects of mutations on the experimentally determined SH3 binding energetics, within the uncertainties of the measurements, indicating that conformational fluctuations in SH3, and in particular the RT loop region, are structurally diverse and are well-approximated by the randomly configured states. The mutated positions in SH3 were distant to the binding site, and involved Ala and Gly substitutions of solvent exposed positions in the RT loop. To characterize these fluctuations, we applied principal coordinate analysis to the computed ensembles, uncovering the principal modes of conformational variation. It is shown that the observed differences in binding affinity between each mutant, and thus the apparent coupling between the mutated sites, can be described in terms of the changes in these principal modes. These results indicate that dynamic loops in proteins can populate a broad conformational ensemble, and that a quantitative understanding of molecular recognition requires consideration of the entire distribution of states.Protein conformational fluctuations are essential for biological function 1 , and perturbations to fluctuations brought about by mutation or environmental changes are known to affect proteins functionally 2 . Despite clear experimental evidence for a dependence of function on fluctuations 3,4,5 , the impact of conformational heterogeneity on important biological processes, such as substrate:ligand recognition, remains poorly understood 3,6 . Clearly, methods that can relate the functional and structural character of proteins are needed to understand mechanistically, and quantitatively, the role that fluctuations have in biological function.To investigate the impact of structural fluctuations on molecular recognition, we selected the C-terminal src homology 3 (SH3) domain of the C. Elegans protein SEM5 (SEM5 C-SH3). SH3 domains are a conserved structural motif found in many regulatory proteins 7 . The typical function of these domains is to mediate protein:protein interactions in signaling pathways by *Corresponding author: E-mail: vjhilser@utmb.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript recognizing proline-rich sequences that adopt a left-handed polyproline II helical structure 8, 9,10,11 ...

Langevin dynamics is used to compute the time evolution of the nonequilibrium motion of the atomic coordinates of a protein in response to ligand dissociation. The protein potential energy surface (PES) is approximated by a harmonic basin about the minimum of the unliganded state. Upon ligand dissociation, the protein undergoes relaxation from the bound to the unbound state. A coarse graining scheme based on rotation translation blocks (RTB) is applied to the relaxation of the two domain iron transport protein, ferric binding protein. This scheme provides a natural and efficient way to freeze out the small amplitude, high frequency motions within each rigid fragment, thereby allowing for the number of dynamical degrees of freedom to be reduced. The results obtained from all flexible atom (constraint free) dynamics are compared to those obtained using RTB-Langevin dynamics. To assess the impact of the assumed rigid fragment clustering on the temporal relaxation dynamics of the protein molecule, three distinct rigid block decompositions were generated and their responses compared. Each of the decompositions was a variant of the one-block-per-residue grouping, with their force and friction matrices being derived from their fully flexible counterpart. Monitoring the time evolution of the distance separating a selected pair of amino acids, the response curves of the blocked decompositions were similar in shape to each other and to the control system in which all atomic degrees of freedom are fully independent. The similar shape of the blocked responses showed that the variations in grouping had only a minor impact on the kinematics. Compared with the all atom responses, however, the blocked responses were faster as a result of the instantaneous transmission of force throughout each rigid block. This occurred because rigid blocking does not permit any intrablock deformation that could store or divert energy. It was found, however, that this accelerated response could be successfully corrected by scaling each eigenvalue in the appropriate propagation matrix by the least-squares fitted slope of the blocked vs nonblocked eigenvalue spectra. The RTB responses for each test system were dominated by small eigenvalue overdamped Langevin modes. The large eigenvalue members of each response dissipated within the first 5 ps, after which the long time response was dominated by a modest set of low energy, overdamped normal modes, that were characterized by highly cooperative, functionally relevant displacements. The response assuming that the system is in the overdamped limit was compared to the full phase space Langevin dynamics results. The responses after the first 5 ps were nearly identical, confirming that the inertial components were significant only in the initial stages of the relaxation. Since the propagator matrix in the overdamped formulation is real-symmetric and does not require the inertial component in the propagator, the computation time and memory footprint was reduced by 1 order of magnitude.

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