High-resolution terahertz spectroscopy of crystalline trialanine: extreme sensitivity to beta-sheet structure and cocrystallized water

High-resolution terahertz absorption spectra (0.06-3 THz) have been obtained at 4.2 K for three crystalline forms of trialanine [H2+-(Ala)3-O-]. The crystal structures differ in their beta-sheet forms (parallel vs antiparallel) and in their water composition (hydrated vs dehydrated antiparallel beta-sheet). The spectra are nearly vibrationally resolved, with little absorption below 1 THz. In sharp contrast to observations made in the mid-IR region, the spectral patterns of all three forms are qualitatively different, illustrating the extreme sensitivity to changes in the intermolecular hydrogen-bonding networks that stabilize peptide crystals. Predictions obtained from a classical force field model (CHARMM) and density functional theory (DFT/PW91) for periodic solids are compared with the X-ray structural data and the terahertz absorption spectra. In general, the results for the parallel beta-sheet are in better agreement with experiment than those for the antiparallel beta-sheet. For all three structures, however, most hydrogen bond distances are underestimated at both levels of theory, and the predicted absorption features are significantly red-shifted for the two antiparallel beta-sheet structures. Moreover, the nuclear motions predicted at the two levels of theory are qualitatively different. These results indicate that the PW91 functional is not sufficient to treat the weak intersheet hydrogen bonding present in the different beta-sheet forms and strongly suggest the need for improved force field models that include three-atom hydrogen-bonding terms for periodic solids.     

Functional concerted motions in the bovine serum retinol-binding protein

The large concerted motions in the apo/holo bovine serum retinol- binding protein were studied using molecular dynamics simulation and 'essential dynamics' analysis. Initially, concerted motions were calculated from conformational differences between various crystal structures. The dynamic behaviour of the protein in the configurational space directions, described by these concerted motions, is analysed. This reveals that the large backbone dynamics of the protein is not influenced by the presence of retinol. Study of free retinol dynamics and retinol in the retinol binding site reveals that the protein binds retinol in a favourable conformation, as opposed to what has been previously described for the bovine cellular retinol-binding protein.     

Template-directed assembly of a de novo designed protein

Many naturally occurring biomaterials are composed of laminated structures in which layers of beta-sheet proteins alternate with layers of inorganic mineral. These ordered laminates often have structural and mechanical properties that differ significantly from those of nonbiological materials. An important step in the construction of novel biomaterials is the creation of composites wherein a de novo designed protein assembles into an ordered structure. To achieve this goal, we layered a de novo protein onto the surface of highly ordered pyrolytic graphite (HOPG). The protein was derived from a combinatorial library of novel sequences designed to fold into amphiphilic beta-sheet structures. Atomic force microscopy reveals that the protein assembles on the HOPG surface into ordered fibers aligned in three orientations at 120 degrees to each other. The symmetry and extent of the ordered regions indicate that the hexagonal lattice underlying the graphite surface templates assembly of millions of protein molecules into a highly ordered structure.     

The promiscuity of beta-strand pairing allows for rational design of beta-sheet face inversion

Recent studies suggest the dominant role of main-chain H-bond formation in specifying beta-sheet topology. Its essentially sequence-independent nature implies a large degree of freedom in designing beta-sheet-based nanomaterials. Here we show rational design of beta-sheet face inversions by incremental deletions of beta-strands from the single-layer beta-sheet of Borrelia outer surface protein A. We show that a beta-sheet structure can be maintained when a large number of native contacts are removed and that one can design large-scale conformational transitions of a beta-sheet such as face inversion by exploiting the promiscuity of strand-strand interactions. High-resolution X-ray crystal structures confirmed the success of the design and supported the importance of main-chain H-bonds in determining beta-sheet topology. This work suggests a simple but effective strategy for designing and controlling nanomaterials based on beta-rich peptide self-assemblies.     

An oligopeptide containing the C-terminal sequence of RNase a has a potent RNase a binding property

We demonstrate that an oligopeptide containing the C-terminal sequence of RNase A binds to RNase A in a stoichiometric and site-specific manner. Our observations are consistent with the interaction found in the major domain-swapped RNase A dimer, so that the peptide binding may be promoted through the swapping with the C-terminal beta-sheet of RNase A. Because the design of a protein-binding peptide is much simpler than other methods such as the combinatorial method, we propose that investigation using an oligopeptide may be of general application to domain swapping in proteins as well as for the development of an oligopeptide tool that specifically binds to a target protein.     

Supramolecular bidentate ligands by metal-directed in situ formation of antiparallel beta-sheet structures and application in asymmetric catalysis

The principles of protein structure design, molecular recognition, and supramolecular and combinatorial chemistry have been applied to develop a convergent metal-ion-assisted self-assembly approach that is a very simple and effective method for the de novo design and the construction of topologically predetermined antiparallel beta-sheet structures and self-assembled catalysts. A new concept of in situ generation of bidentate P-ligands for transition-metal catalysis, in which two complementary, monodentate, peptide-based ligands are brought together by employing peptide secondary structure motif as constructing tool to direct the self-assembly process, is achieved through formation of stable beta-sheet motifs and subsequent control of selectivity. The supramolecular structures were studied by (1)H, (31)P, and (13)C NMR spectroscopy, ESI mass spectrometry, X-ray structure analysis, and theoretical calculations. Our initial catalysis results confirm the close relationship between the self-assembled sheet conformations and the catalytic activity of these metallopeptides in the asymmetric rhodium-catalyzed hydroformylation. Good catalyst activity and moderate enantioselectivity were observed for the selected combination of catalyst and substrate, but most importantly the concept of this new methodology was successfully proven. This work presents a perspective interface between protein design and supramolecular catalysis for the design of beta-sheet mimetics and screening of libraries of self-organizing supramolecular catalysts.     

Weak long-range correlated motions in a surface patch of ubiquitin involved in molecular recognition

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 ? in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogen-bonding network.     

Dynamics of a protein and water molecules surrounding the protein: hydrogen-bonding between vibrating water molecules and a fluctuating protein

Internal and rigid-body motions of bovine pancreatic trypsin inhibitor (BPTI) and of water molecules surrounding the BPTI are studied in a vicinity of an energy minimum using a normal mode analysis proposed as the independent molecule model. Water's rigid-body motion is predominant in comparison to its internal motions. We have derived information about the relationship between the magnitude of a thermal ellipsoid of an H-bonding atom and the anisotropy of its ellipsoid, and the relationship between the magnitude of the ellipsoid and the H-bond strength. We see a relationship between vibrational frequencies (assuming rigid-body motion of the water molecules) and the H-bond strength of the water taking part in this H-bonding. Analyzing the H-bond strength, we found that a hydrogen in water is likely to H-bond to oxygen in the protein, whereas an oxygen in water has a less strong preference to H-bond to the protein. For water molecules acting as the hydrogen acceptor, strong H-bonding has longer lifetimes than weak H-bonding.     

Molecular motion in crystalline naphthalene: analysis of multi-temperature X-ray and neutron diffraction data

Single crystals of h8-naphthalene have been examined by both X-ray and neutron diffraction over a range of temperatures from 5 to 295 K. The aim of this case study was to measure the anisotropic displacement parameters (ADPs) of carbons and hydrogens and to interpret them using the model of thermal motion proposed by Bürgi and Capelli (Acta Cryst. 2000, A56, 403). The traditional rigid-body analysis expresses the low-frequency motions in terms of molecular translations and librations only, whereas the Bürgi-Capelli treatment also includes the high-frequency internal modes. We show that a considerable improvement occurs by representing the internal modes by a single second-rank tensor and that a further improvement follows by including a Grüneisen parameter to account for volume thermal expansion. By applying the treatment to multi-temperature diffraction data, there is a considerable reduction in the ratio of number of adjustable parameters/number of independent observations.     

Surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) brushes as templates for enzyme immobilization

We explored surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brushes as potential templates for protein immobilization. The brushes were grown using atom transfer radical polymerization from surface-anchored initiators and characterized by a combination of ellipsometry, atomic force microscopy, and X-ray photoelectron spectroscopy. RNase A was immobilized as a model enzyme through the nucleophilic attack of azlactone by the amine groups in the lysines located in the protein. The surface density of RNase A increased linearly from 5 to 50 nm. For 50 nm thick poly(2-vinyl-4,4-dimethyl azlactone) brushes, 7.5 microg/cm2 of RNase A was bound. The kinetics and thermodynamics of RNase A immobilization, the activity relative to surface density, and the pH and temperature dependence were examined. A Langmuir-like model for binding kinetics indicates that the kinetics are controlled by the rate of adsorption of RNase A and has an adsorption rate constant, k(ads), of 2.8 x 10(-8) microg(-1) s(-1) cm3. A maximum relative activity of approximately 0.95, which is near the activity of free RNase A, was reached at 1.2 microg/cm2 (approximately 3.0 monolayers) of immobilized RNase A. The immobilized RNase A had a similar temperature and pH dependence as free RNase A, indicating no significant change in conformation. The PVDMA template was extended to other biotechnologically relevant enzymes, such as deoxyribonuclease I, glucose oxidase, glucoamylase, and trypsin, with relative activities higher than or comparable to those of enzymes immobilized by other means. PVDMA brushes offer an efficient route to immobilize proteins via the ring opening of azlactone without the need for activation or pretreatment while retaining high relative activities of the bound enzymes.     

Hydrogen-bonded OH stretching modes of methanol clusters: a combined IR and Raman isotopomer study

A comprehensive study of the OH and OD stretching fundamentals in clusters of methanol and its isotopomers CH(3)OD, CD(3)OH, and CD(3)OD provides detailed insights into the hydrogen-bond mediated coupling as a function of cluster size. The combination of infrared and Raman supersonic jet spectroscopy enables the observation and assignment of all hydrogen-bonded OH stretching modes of isolated methanol trimer and methanol tetramer. A consistent explanation for the spectral complexity observed more than a decade ago in methanol trimer in terms of low-frequency methyl umbrella motions is provided. Previous explanations based on cluster isomerism or anharmonic resonances are ruled out by dedicated jet experiments. The first experimental lower bound for concerted quadruple proton transfer in S(4) symmetric methanol tetramer is derived and compared with theoretical predictions. The observed isotope effects offer insights into the anharmonicity of the localized OH bond. The performance of harmonic B3LYP and MP2 calculations in predicting hydrogen-bond-induced spectral shifts and couplings is investigated.     

Protein motions promote catalysis

A relationship between molecular dynamics motions of noncatalytic residues and enzyme activity has recently been proposed. We present examples where mutations either near or distal from the active site residues modify internal enzyme motion with resulting modification of catalysis. A better understanding of internal protein motions correlated to catalysis will lead to a greater insight into enzyme function.     

Differentiation of beta-sheet-forming structures: ab initio-based simulations of IR absorption and vibrational CD for model peptide and protein beta-sheets.

Ab initio quantum mechanical computations of force fields (FF) and atomic polar and axial tensors (APT and AAT) were carried out for triamide strands Ac-A-A-NH-CH(3) clustered into single-, double-, and triple-strand beta-sheet-like conformations. Models with phi, psi, and omega angles constrained to values appropriate for planar antiparallel and parallel as well as coiled antiparallel (two-stranded) and twisted antiparallel and parallel sheets were computed. The FF, APT, and AAT values were transferred to corresponding larger oligopeptide beta-sheet structures of up to five strands of eight residues each, and their respective IR and vibrational circular dichroism (VCD) spectra were simulated. The antiparallel planar models in a multiple-stranded assembly give a unique IR amide I spectrum with a high-intensity, low-frequency component, but they have very weak negative amide I VCD, both reflecting experimental patterns seen in aggregated structures. Parallel and twisted beta-sheet structures do not develop a highly split amide I, their IR spectra all being similar. A twist in the antiparallel beta-sheet structure leads to a significant increase in VCD intensity, while the parallel structure was not as dramatically affected by the twist. The overall predicted VCD intensity is quite weak but predominantly negative (amide I) for all conformations. This intrinsically weak VCD can explain the high variation seen experimentally in beta-forming peptides and proteins. An even larger variation was predicted in the amide II VCD, which had added complications due to non-hydrogen-bonded residues on the edges of the model sheets.     

Exploring multiple timescale motions in protein GB3 using accelerated molecular dynamics and NMR spectroscopy

Biological function relies on the complex spectrum of conformational dynamics occurring in biomolecules. We have combined Accelerated Molecular Dynamics (AMD) with experimental results derived from NMR to probe multiple time-scale motions in the third IgG-binding domain of Protein G (GB3). AMD is shown to accurately reproduce the amplitude and distribution of slow motional modes characterized using residual dipolar couplings, reporting on dynamics up to the millisecond timescale. In agreement with experiment, larger amplitude slower motions are localized in the beta-strand/loop motif spanning residues 14-24 and in loop 42-44. Principal component analysis shows these fluctuations participating in the primary mode, substantiating the existence of a correlated motion traversing the beta-sheet that culminates in maximum excursions at the active site of the molecule. Fast dynamics were simulated using extensive standard MD simulations and compared to order parameters extracted from 15N relaxation. Notably 60 2-ns fully solvated MD simulations exploring the different conformational substates sampled from AMD resulted in better reproduction of order parameters compared to the same number of simulations starting from the relaxed crystal structure. This illustrates the inherent dependence of protein dynamics on local conformational topology. The results provide rare insight into the complex hierarchy of dynamics present in GB3 and allow us to develop a model of the conformational landscape native to the protein, appearing as a steep sided potential well whose flat bottom comprises multiple similar but discrete conformational substates.     

Proton-coupled electron transfer in solution, proteins, and electrochemistry

Recent advances in the theoretical treatment of proton-coupled electron transfer (PCET) reactions are reviewed. These reactions play an important role in a wide range of biological processes, as well as in fuel cells, solar cells, chemical sensors, and electrochemical devices. A unified theoretical framework has been developed to describe both sequential and concerted PCET, as well as hydrogen atom transfer (HAT). A quantitative diagnostic has been proposed to differentiate between HAT and PCET in terms of the degree of electronic nonadiabaticity, where HAT corresponds to electronically adiabatic proton transfer and PCET corresponds to electronically nonadiabatic proton transfer. In both cases, the overall reaction is typically vibronically nonadiabatic. A series of rate constant expressions have been derived in various limits by describing the PCET reactions in terms of nonadiabatic transitions between electron-proton vibronic states. These expressions account for the solvent response to both electron and proton transfer and the effects of the proton donor-acceptor vibrational motion. The solvent and protein environment can be represented by a dielectric continuum or described with explicit molecular dynamics. These theoretical treatments have been applied to numerous PCET reactions in solution and proteins. Expressions for heterogeneous rate constants and current densities for electrochemical PCET have also been derived and applied to model systems.     

Functionally relevant protein motions: extracting basin-specific collective coordinates from molecular dynamics trajectories

Functionally relevant motion of proteins has been associated with a number of atoms moving in a concerted fashion along so-called "collective coordinates." We present an approach to extract collective coordinates from conformations obtained from molecular dynamics simulations. The power of this technique for differentiating local structural fluctuations between classes of conformers obtained by clustering is illustrated by analyzing nanosecond-long trajectories for the response regulator protein Spo0F of Bacillus subtilis, generated both in vacuo and using an implicit-solvent representation. Conformational clustering is performed using automated histogram filtering of the inter-C(alpha) distances. Orthogonal (varimax) rotation of the vectors obtained by principal component analysis of these interresidue distances for the members of individual clusters is key to the interpretation of collective coordinates dominating each conformational class. The rotated loadings plots isolate significant variation in interresidue distances, and these are associated with entire mobile secondary structure elements. From this we infer concerted motions of these structural elements. For the Spo0F simulations employing an implicit-solvent representation, collective coordinates obtained in this fashion are consistent with the location of the protein's known active sites and experimentally determined mobile regions.     

The organization and assembly of a beta-sheet formed by a prion peptide in solution: an isotope-edited FTIR study

Insight into the details of protein misfolding diseases requires a detailed understanding of the conformation and dynamics of multistrand beta-sheet aggregates. Here, we report an isotope-edited FTIR study of a model peptide directed at the elucidation of residue-level details of the structure and mechanism of a beta-sheet aggregate. A series of specifically isotope-labeled derivatives of a short peptide (H1) derived from residues 109 through 122 of the prion protein PrPC have been synthesized and characterized by FTIR. On the basis of the analysis of variable temperature FTIR spectra of these peptides in solution, the organization of strands within the beta-sheets has been determined; at equilibrium, the strands form a beta-sheet in which the hydrophobic core (112-122) participates in the sheet structure, resulting in the alignment of residue 117 in all of the strands. The peptides initially form a kinetically trapped intermediate beta-sheet, with a distribution of strand alignments, which can be rearranged into the stable equilibrium conformation by an annealing cycle. These observations are discussed in terms of the biological significance of residue 117 of the prion protein and the mechanism of beta-aggregate nucleation in prion proteins.     

Rapid determination of ribonuclease and microanalysis of heparin with a SAW/conductance sensor

A new method using a surface acoustic wave (SAW)/conductance sensor has been described in this paper for rapid determination of ribonuclease (RNase) and microanalysis of heparin. The assay of RNase is based on the change in conductance of the solution caused by enzymatic reaction between ribonucleic acid (RNA) and RNase and the analysis of heparin is based on its inhibitory action on RNase. A linear relationship between frequency response and enzyme concentration is obtained and the detection limit of RNase is evaluated to be 0.17 mug ml(-1). The recovery of the sensor system ranges from 95.8 to 105.0%. The inhibition of heparin is a competitive one and the possible inhibition mechanism is discussed. The kinetic parameters and inhibition parameters are estimated. The calibration graph is rectilinear for 相似文献   

Viscoelasticity of gelatin surfaces probed by AFM noise analysis

The viscoelastic properties of surfaces of swollen gelatin were investigated by analyzing the Brownian motion of an atomic force microscopy (AFM) cantilever in contact with the gel surface. A micron-sized glass sphere attached to the AFM cantilever is used as the dynamic probe. When the sphere approaches the gelatin surface, there is a static repulsive force without a jump into contact. The cantilever's Brownian movement is monitored in parallel, providing access to the dynamic sphere-surface interaction as quantified by the dynamic spring constant, kappa, and the drag coefficient, xi. Gelatin is used as a model substance for a variety of other soft surfaces, where the stiffness of the gel can be varied via the solvent quality, the bloom number, and the pH. The modulus derived from the static force-distance curve is in the kPa range, consistent with the literature. However, the dynamic spring constant as derived from the Brownian motion is much larger than the static differential spring constant dF/dz. On retraction, one observes a rather strong adhesion hysteresis. The strength of the bridge (as given by the dynamic spring constant and the drag coefficient) is very small.     

Model for a three-center decomposition reaction. I. Perfluorodiazirine

A semiempirical approach has been used to evaluate rate parameters for a three-center decomposition reaction from the point of view of transition state theory, with perfluorodiazirine serving as the prototype molecule. Several activated complex models are considered in which the reaction coordinate is chosen as the ? NCN bending mode. The constraints imposed include the principle of concerted bond-order conservation in passing from the initial to the final state, and use is made of empirical bond order–bond length and bond order–force constant relationships. The geometric configuration of the transition state sought is one which conforms with the lowest energy path and is also consistent with the observed entropy of activation. The potential energy of activation is taken as the optimum difference in binding energies (based on the INDO method) between the transition and initial states, and the critical energy is obtained by applying a correction for the zero-point energy difference, derived from normal coordinate analysis. Satisfactory agreement is found in the case of the activated complex model for which the total bond order is conserved and bonds undergoing rupture are assigned a fractional bond order (FBO) of 2/3, derived from the postulate (FBO) = α/β whe re α(=2) is the number of bonds breaking, and β(=3) is the number of bonds undergoing change in the ring opening.