Dynamics of water confined in the interdomain region of a multidomain protein

Molecular dynamics simulations are performed to study the dynamics of interfacial water confined in the interdomain region of a two-domain protein, BphC enzyme. The results show that near the protein surface the water diffusion constant is much smaller and the water-water hydrogen bond lifetime is much longer than that in bulk. The diffusion constant and hydrogen bond lifetime can vary by a factor of as much as 2 in going from the region near the hydrophobic domain surface to the bulk. Water molecules in the first solvation shell persist for a much longer time near local concave sites than near convex sites. Also, the water layer survival correlation time shows that on average water molecules near the extended hydrophilic surfaces have longer residence times than those near hydrophobic surfaces. These results indicate that local surface curvature and hydrophobicity have a significant influence on water dynamics.     

A model calculation of protonic mobility in ice

The protonic conductivity in ice crystals is considered within the framework of strong coupling developed earlier by the present authors for semiconductors with narrow bands and strong coupling of electrons with phonons. The generalization for a two-band model is developed. The model used is the one described by Gosar and Pintar, the principal feature of which is the concept of the protonic conduction bands. The analytical expression for the protonic mobility is given. The results of the theory are in qualitative agreement with the experimental data.     

Solid-state NMR on a large multidomain integral membrane protein: the outer membrane protein assembly factor BamA

Multidomain proteins constitute a large part of prokaryotic and eukaryotic proteomes and play fundamental roles in various physiological processes. However, their structural characterization is challenging because of their large size and intrinsic flexibility. We show here that motional-filtered high-resolution solid-state NMR (ssNMR) experiments allow for the observation and structural analysis of very large multidomain membrane proteins that are characterized by different motional time scales. This approach was used to probe the folding of the 790-residue membrane protein BamA, which is the core component of the Escherichia coli outer membrane protein assembly machinery. A combination of dipolar- and scalar-based two-dimensional ssNMR experiments applied to two uniformly (13)C,(15)N-labeled BamA variants revealed characteristic secondary structure elements and distinct dynamics within the BamA transmembrane protein segment and the periplasmic POTRA domains. This approach hence provides a general strategy for collecting atomic-scale structural information on multidomain (membrane) proteins in a native-like environment.     

Mutational effects on protein structural changes and interdomain interactions in the blue-light sensing LOV protein YtvA

Mutagenesis studies on the phototropin-related protein YtvA from Bacillus subtilis have revealed the role of selected structural elements in interdomain communication. The LOV (light, oxygen, voltage) domain of YtvA undergoes light-driven reactions similar to that of phot-LOV, with reversible formation of a covalent flavin-cysteine adduct. The mutated proteins Ytva-E105L and YtvA-E56Q have been studied by UV fluorescence and circular dichroism (CD) spectroscopy. E105 (L in phototropin) is located at the solvent-exposed surface of the LOV domain central beta-sheet, demonstrated to participate in interdomain interaction in phototropin. CD data show that YtvA-E105L has a lower alpha-helix content in the dark and undergoes larger light-driven conformational changes than YtvA-WT. The E56Q mutation breaks the E56-K97 salt bridge, a structural element highly conserved within the LOV series. In YtvA-E56Q the CD spectrum is the same as in YtvA-WT, although the conserved W103 becomes more exposed to the solvent and the dark-recovery kinetics is slower. These results indicate that the E56-K97 salt bridge stabilizes locally the protein structure and participates in the regulation of the photocycle but has negligible effects on the overall structure. The E105L mutation, instead, highlights the involvement of the central beta-sheet in the light-driven conformational changes in LOV proteins.     

A simple model of the high-frequency dynamic mobility in concentrated suspensions

Because electroacoustic techniques are gaining interest in many fields of colloid science, a number of theories dealing with the phenomenon of electrophoresis in high-frequency (on the order of the MHz) electric fields have been developed. In the present work we propose a straightforward derivation of a simple formula for the dynamic mobility of colloidal particles in mildly concentrated systems. Starting with a simple expression for the electrophoretic mobility in dilute suspensions, given as a function of the zeta potential and of the dipole coefficient, we introduce successive corrections related to: (i) the back flow of fluid induced by the electrophoretic motion of the particles; (ii) the electrostatic interactions among particles; (iii) the difference between the macroscopic and the external electric fields; (iv) the difference between the zero-momentum and the laboratory reference frames. Considering furthermore that the frequency dependence of the dipole coefficient is due to the Maxwell-Wagner-O'Konski double-layer relaxation, we obtain a mobility expression that compares well with other (semi)analytical models and (in proper conditions) with numerical cell-model calculations. However, its main merit is that it allows to understand, to a large extent, the physical origin of the frequency and volume fraction dependences of the dynamic mobility.     

Tension and mobility of a DNA fragment in the lakes-straits model

We consider numerically, in the framework of the lakes-straits model, the mobility of a DNA chain under field-inversion gel-electrophoresis (FIGE). Here we investigate the role of the gel's structure on the mobility. We consider two situations: (i) a DNA chain fragment is bounded by two straits (narrow gateways) at a fixed distance, but is otherwise free, and (ii) the fragment is, in addition, confined with in a closed volume (pore). We calculate the tension on the DNA fragment in the two cases. For Gaussian chains we evaluate the corresponding statistical weights exactly, using a cellular automation algorithm. We find that the resulting tension differs drastically in the two models considered. Nonetheless this difference influences only weakly the overall FIGE mobility.     

Proline hydroxylation alters the electrophoretic mobility of pulmonary surfactant-associated protein A

Studies from several laboratories involving amino acid analysis and sequencing of the Mr 35,000 pulmonary surfactant-associated proteins (SP-A) have detected hydroxyproline residues. These residues are present in a region with a collagen-like sequence that has been revealed by direct amino acid sequencing and from the deduced amino acid sequence of the cDNA clones coding for SP-A. We treated human lung tissue with tunicamycin to block N-glycosylation and with 2,2-dipyridyl to inhibit the hydroxylation of proline residues. The SP-A synthesized under these conditions showed a shift in apparent molecular weight to 27,000 and 29,000 compared to 29,000 and 31,000 for SP-A synthesized in the presence of tunicamycin alone. Dipyridyl treatment alone caused an alteration in electrophoretic mobility similar to that seen with tunicamycin, although this was more difficult to evaluate since changes in molecular weight due to glycosylation occurred under these conditions. These results indicate that proline hydroxylation in the collagen-like portion of SP-A decreases its electrophoretic mobility.     

A voltammetric study of interdomain electron transfer within sulfite oxidase

Protein film voltammetry of chicken liver sulfite oxidase (SO) bound at the pyrolytic graphite "edge" or modified gold electrodes shows that catalytic electron transport is controlled by the inherent electrochemical characteristics of the heme b domain and conformational changes that allow intramolecular electron transfer with the molybdenum active site. In the absence of sulfite, a single nonturnover electrochemical signal is observed at +90 mV (vs SHE) that is assigned to heme b. In the presence of sulfite, this signal transforms into a catalytic wave at similar potential. The shape and negligible pH dependence of this wave indicate that catalytic turnover is controlled by the one-electron transfers through heme b. The smaller turnover numbers obtained in this experiment (k(cat) approximately 2-4 s(-1), as compared to 100 s(-1) in solution) suggest that only a small fraction of SO is bound at the electrode in a manner that permits the conformational change necessary for fast interdomain electron transfer.     

Hormone receptor mobility and catecholamine binding in membranes. A theoretical model.

[3H]-Catecholamine binding to intact cells, isolated cell membranes, and to several isolated macromolecules has been shown by several laboratories to be neither stereospecific nor inhibited by known beta-antagonists. Since additional evidence indicates that this binding is not an artifact (i.e. due neither to the binding of a catecholamine oxidation product nor hormone binding to a catabolic enzyme such as COMT), the question remains as to whether this represents binding to a bona fide membrane receptor. Because all ligands which bind strongly or compete for this binding possess a catechol group, one possible explanation is that the binding affinity is primarily determined by the catechol moiety, whereas the correct stereoisomer of the side chain is necessary to activate the receptor. Thus, although binding is a necessary condition for hormone action, the necessary and sufficient condition for activation of adenyl cyclase is both the catechol group and the correct stereoisomer of the side chain. A theoretical model is developed here to provide a quantitative basis for this hypothesis. This model extends the current concept of distinct subunits in the adenyl cyclase system by separating the receptors from the catalytic sites and placing them at separate locations within the membrane. Utilizing the spare receptor model of Furchgott, and the mobility of macromolecules within a "lipid sea," the appropriate equations to predict both hormone binding and enzyme activation are derived. Using the observed affinity constants from catecholamine binding studies, it is then shown that this model can predict the experimental observation and hence explain the apparent dichotomy arising from binding enzyme activation studies.     

Conformer selection of protein ions by ion mobility in a triple quadrupole mass spectrometer

Electrospray mass spectra of multiply charged protein molecules show two distinct charge state distributions proposed to correspond to a more highly charged, open conformational form and a lower charged, folded form. Elastic collisions carried out in the radiofrequency-only collision cell of a triple quadrupole mass spectrometer have dramatic effects on the appearance of the mass spectra. The different cross sectional areas of the conformers allow preferential selection of one charge state distribution over the other on the basis of ion mobility. Preferential selection is dependent on the nature and pressure of the target gas as well as the nature of the protein. In the case of positively charged horse heart apomyoglobin (MW 16,951 da), a high charge state distribution centered around (M + 20H)²⁰⁺ predominates at low target gas pressures and a second distribution centered around (M + 10H)¹⁰⁺ predominates at high target gas pressures. Bimodal distributions are observed at intermediate pressures and, remarkably, charge states between the two distributions are not effectively populated under most of the conditions examined. Hard sphere collision calculations show large differences in collision frequencies and in the corresponding kinetic energy losses for the two conformational states and they demonstrate that the observed charge state selectivity can be explained through elastic collisions.     

Refinement of multidomain protein structures by combination of solution small-angle X-ray scattering and NMR data

Determination of the 3D structures of multidomain proteins by solution NMR methods presents a number of unique challenges related to their larger molecular size and the usual scarcity of constraints at the interdomain interface, often resulting in a decrease in structural accuracy. In this respect, experimental information from small-angle scattering of X-ray radiation in solution (SAXS) presents a suitable complement to the NMR data, as it provides an independent constraint on the overall molecular shape. A computational procedure is described that allows incorporation of such SAXS data into the mainstream high-resolution macromolecular structure refinement. The method is illustrated for a two-domain 177-amino-acid protein, gammaS crystallin, using an experimental SAXS data set fitted at resolutions from approximately 200 A to approximately 30 A. Inclusion of these data during structure refinement decreases the backbone coordinate root-mean-square difference between the derived model and the high-resolution crystal structure of a 54% homologous gammaB crystallin from 1.96 +/- 0.07 A to 1.31 +/- 0.04 A. Combining SAXS data with NMR restraints can be accomplished at a moderate computational expense and is expected to become useful for multidomain proteins, multimeric assemblies, and tight macromolecular complexes.     

Segmental mobility model for diffusion of gases in polymers

Diffusion of small molecules in polymers is described quantitatively in terms of segmental mobility processes. The diffusion coefficient depends on a diffusive jump length, which is characteristic of the polymer, and a jump frequency, which is equated to the segmental mobility rate. The presence of a particular solute increases mobility of the surrounding polymer segments by a predictable amount, which is related to the partial molar volume of the solute. The theory is fit to experimental diffusion data, and partial molar volumes are calculated from the fitting parameters. Good agreement with experimental partial molar volumes is obtained.     

Structural assembly of multidomain proteins and protein complexes guided by the overall rotational diffusion tensor

We present a simple and robust approach that uses the overall rotational diffusion tensor as a structural constraint for domain positioning in multidomain proteins and protein-protein complexes. This method offers the possibility to use NMR relaxation data for detailed structure characterization of such systems provided the structures of individual domains are available. The proposed approach extends the concept of using long-range information contained in the overall rotational diffusion tensor. In contrast to the existing approaches, we use both the principal axes and principal values of protein's rotational diffusion tensor to determine not only the orientation but also the relative positioning of the individual domains in a protein. This is achieved by finding the domain arrangement in a molecule that provides the best possible agreement with all components of the overall rotational diffusion tensor derived from experimental data. The accuracy of the proposed approach is demonstrated for two protein systems with known domain arrangement and parameters of the overall tumbling: the HIV-1 protease homodimer and Maltose Binding Protein. The accuracy of the method and its sensitivity to domain positioning are also tested using computer-generated data for three protein complexes, for which the experimental diffusion tensors are not available. In addition, the proposed method is applied here to determine, for the first time, the structure of both open and closed conformations of a Lys48-linked diubiquitin chain, where domain motions render impossible accurate structure determination by other methods. The proposed method opens new avenues for improving structure characterization of proteins in solution.     

Extended model free approach to analyze correlation functions of multidomain proteins in the presence of motional coupling

Interdomain motion in proteins plays an important role in biomolecular interaction. Its presence also complicates interpretation of many spectroscopy measurements. Nuclear magnetic resonance (NMR) study of domain dynamics relies on knowledge of its rotational correlation function. The extended model free (EMF) approach has been implemented to analyze coupled domain and overall motions for calmodulin, a dual-domain protein; however, the validity of EMF treatment in coupled motion has not been tested. We performed stochastic simulations on a dual-vector system employing two simple restraints to drive hydrodynamics and domain coupling: (1) both unitary vectors diffuse randomly on the surface of a sphere and (2) vectors are correlated through user-defined intervector potential. The resulting correlation curve can be adequately fit with either a single- or double-exponential decay function. The latter is consistent with the EMF treatment. The derived order parameters S (2) range from about 0.4 to 1, while the motion separation, the ratio of overall and domain motion time scales (tau m/tau s), ranges from 1 to 4. A complete overlap between time scales occurs when S (2) is less than 0.4, and the correlation function effectively behaves as a single-exponential. The S (2) values are consistent with theoretical predictions from the given potential function, differing by no more than 0.03, suggesting EMF to be a generally valid approach. In addition, from the dependence of S (2) on tau m/tau s obtained from simulation, we found a cosine potential, favoring extended conformers, as opposed to the normally assumed cone potential, reached a better agreement to experimental data.     

A multidisciplinary approach to probing enthalpy-entropy compensation and the interfacial mobility model

In recent years, interfacial mobility has gained popularity as a model with which to rationalize both affinity in ligand binding and the often observed phenomenon of enthalpy-entropy compensation. While protein contraction and reduced mobility, as demonstrated by computational and NMR techniques respectively, have been correlated to entropies of binding for a variety of systems, to our knowledge, Raman difference spectroscopy has never been included in these analyses. Here, nonresonance Raman difference spectroscopy, isothermal titration calorimetry, and X-ray crystallography were utilized to correlate protein contraction, as demonstrated by an increase in protein interior packing and decreased residual protein movement, with trends of enthalpy-entropy compensation. These results are in accord with the interfacial mobility model and lend additional credence to this view of protein activity.     

Temperature dependence of charge mobility in model discotic liquid crystals

We address the calculation of charge carrier mobility of liquid-crystalline columnar semiconductors, a very promising class of materials in the field of organic electronics. We employ a simple coarse-grained theoretical approach and study in particular the temperature dependence of the mobility of the well-known triphenylene family of compounds, combining a molecular-level simulation for reproducing the structural changes and the Miller-Abrahams model for the evaluation of the transfer rates within the hopping regime. The effects of electric field, positional and energetic disorder are also considered. Simulations predict a low energetic disorder (~0.05 eV), slightly decreasing with temperature within the crystal, columnar and isotropic phases, and fluctuations of the square transfer integral of the order of 0.003 eV(2). The shape of the temperature-dependent mobility curve is however dominated by the variation of the transfer integral and barely affected by the disorder. Overall, this model reproduces semi-quantitatively all the features of experimentally measured mobilities, on one hand reinforcing the correctness of the hopping transport picture and of its interplay with system morphology, and on the other suggesting future applications for off-lattice modeling of organic electronics devices.     

Double cushions preserve transmembrane protein mobility in supported bilayer systems

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)-conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 x 10(-8) cm2/s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 x 10(-8) cm2/s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.     

Coupled membrane fluctuations and protein mobility in supported intermembrane junctions

Junctions between lipid membranes make possible cell-free explorations of physical mechanisms that can contribute to protein and lipid organization at a variety of biophysical interfaces. Recent studies of mobile antibodies sandwiched between lipid bilayer membranes have shown that strong intermembrane adhesion and protein mobility alone are sufficient to drive inert proteins into micron-scale patterns of dense and sparse zones. Though the length scale of these patterns was suspected to be related to membrane rigidity, a quantitative understanding has so far been unavailable. We introduce data showing radially structured protein patterns that also demonstrate micron-scale organization. We then provide a simple model that relates the spectrum of membrane fluctuations to the observed protein distributions; in brief, only membrane modes that are slow enough to couple to the protein mobility drive intermembrane protein patterns.