Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins.

The effects of internal motions on residual dipolar NMR couplings of proteins partially aligned in a liquid-crystalline environment are analyzed using a 10 ns molecular dynamics (MD) computer simulation of ubiquitin. For a set of alignment tensors with different orientations and rhombicities, MD-averaged dipolar couplings are determined and subsequently interpreted for different scenarios in terms of effective alignment tensors, average orientations of dipolar vectors, and intramolecular reorientational vector distributions. Analytical relationships are derived that reflect similarities and differences between motional scaling of dipolar couplings and scaling of dipolar relaxation data (NMR order parameters). Application of the self-consistent procedure presented here to dipolar coupling measurements of biomolecules aligned in different liquid-crystalline media should allow one to extract in a "model-free" way average orientations of dipolar vectors and specific aspects of their motions.     

Structural rules for globular proteins.

Is it possible to reach a detailed understanding of the complex three-dimensional structures of native polypeptide chains? In view of the wealth of common physicochemical and phylogenetic features discovered among proteins this question has become reasonable. The current state of discussion is presented in this report.     

Noble-metal nanoparticles directly conjugated to globular proteins

We report the synthesis of gold nanoparticles directly conjugated to bovine serum albumin protein by chemical reduction in aqueous solution. Transmission electron microscopy reveals that the gold nanoparticles are well dispersed with an average diameter less than 2 nm, and elemental analysis verifies the composition of the gold-protein conjugates. Infrared spectroscopy confirms that the polypeptide backbone is not cleaved during the conjugation process and that the side chain functional groups remain intact. Raman spectroscopy demonstrates that the disulfide bonds in the conjugated protein are broken and thus are available for interaction with the nanoparticle surface. This synthesis method is a new technique for directly attaching gold nanoparticles to macromolecular proteins.     

An approach to the core structure of leiodermatolide

The synthesis of the 16-membered core structure of leiodermatolide 40 has been achieved in 26 linear steps starting from (R)-Roche ester. The key steps in the synthesis of 40 are a Stille cross-coupling between two main fragments 11 and 33 having roughly equal size. For the trisubstituted C4/C5 double bond a carbometalation reaction followed by a Suzuki coupling was used. A Yamaguchi macrolactonization furnished macrolactone 39.     

Comparative study of the structure of products of plasma modification of globular and fibrillar proteins

The molecular-mass distribution and amino acid composition of globular (albumin, lysozyme) and fibrillar (collagen) proteins subjected to treatment in electron-beam plasmas of various gases were experimentally studied. The samples were treated in the forms of powders and freeze-dried thin films. The electron-beam plasma treatment of powdered collagen resulted in the formation of low-molecular-mass compounds only. The modification of albumin and lysozyme was accompanied by polymerization of the proteins against the background of insignificant degradation. The plasma-stimulated processes occurred in the surface layer of powder particles, whereas the bulk of the sample remained intact. The degradation and polymerization processes in thin films of globular proteins occurred throughout the entire volume of the sample.     

Quantitation of the global secondary structure of globular proteins by FTIR spectroscopy: Comparison with X-ray crystallographic structure

Fourier transform infrared spectroscopy (FTIR) is potentially a powerful tool for determining the global secondary structure of proteins in solution, providing the spectra are analyzed using a statistically and theoretically justified methodology. We have performed FTIR experiments on 14 globular proteins and two synthetic polypeptides whose X-ray crystal structures are known to exhibit varying types and amounts of secondary structures. Calculation of the component structural elements of the vibrational bands was accomplished using nonlinear regression analysis, by fitting both the amide I and amide II bands of the Fourier self-deconvoluted spectra, the second-derivative spectra, and the original spectra. The methodology was theoretically justified by comparing (via nonlinear regression analysis) the global secondary structure determined after deconvolving into component bands the vibrational amide I envelopes with the calculated structure determined by first principles from Ramachandran analysis of the X-ray crystallographic structure of 14 proteins from the Brookhaven protein data bank. Justification of the nonlinear regression analysis model with respect to experimental and instrumental considerations was achieved by the decomposition of all the bands of benzene and an aqueous solution of ammonium acetate into component bands while floating the Gaussian/Lorentzian character of the line shapes. The results for benzene yield all pure Lorentzian line shapes with no Gaussian character while the ammonium acetate spectra yielded all Gaussian line shapes with no Lorentzian character. In addition, all-protein spectra yielded pure Gaussian line shapes with no Lorentzian character. Finally, the model was statistically justified by recognizing random deviation patterns in the regression analysis from all fits and by the extra sum of squares F-test which uses the degrees of freedom and the root mean square values as a tool to determine the optimum number of component bands required for the nonlinear regression analysis. Results from this study demonstrate that the globular secondary structure calculated from the amide I envelope for these 14 proteins from FTIR is in excellent agreement with the values calculated from the X-ray crystallographic data using three-dimensional Ramachandran analysis, providing that the proper contribution from GLN and ASN side chains to the 1667 and 1650 cm(-1) component bands has been taken into account. The standard deviation of the regression analysis for the per cent helix, extended, turn and irregular conformations was found to be 3.49%, 2.07%, 3.59% and 3.20%, respectively.     

An algebraic operator approach to electronic structure

In this paper, we introduce an algebraic approach to electronic structure calculations. Our approach constructs a Jordan algebra based on the second-quantized electronic Hamiltonian. From the structure factor of this algebra, we show that we can calculate the energy of the ground electronic state of the Hamiltonian operator. We apply our method to several generalized Hubbard models and show that we can usually obtain a significant fraction of the correlation energy for low-to-moderate values of the electronic repulsion parameter while still retaining the O(L(3)) scaling of the Hartree-Fock algorithm. This surprising result, along with several other observations, suggests that our algebraic approach represents a new paradigm for electronic structure calculations which opens up many new directions for research.     

An approach to automated partial structure expansion

An algorithm (ASSEMBLE) to construct all structures consistent with the structural implications of the chemical and spectroscopic properties of an unknown molecule is described. The design of ASSEMBLE takes cognizance of the need to supply some nonoverlapping substructure information in addition to the molecular formula, and the use of structural constraints that cannot be directly expressed as non-overlapping fragments. ASSEMBLE employs several heuristics (rules) intended to avoid the assembly of identical (isomorphic) graphs. To provide a non-redundant list of structures, duplicate structures are recognized and removed by a naming algorithm. ASSEMBLE also perceives different π-resonance forms as identical structures even when they are topologically non-equivalent.     

A quantitative reconstruction of the amide I contour in the IR spectra of globular proteins: from structure to spectrum

The Amide I contours of six globular proteins of varied secondary structure content along with a peptide model for collagen and pulmonary surfactant protein C have been simulated very closely by using a modified GF matrix method. The starting point for the method uses the three-dimensional structure as obtained from the Protein Data Bank. Elements of the interactions between peptide groups (e.g., transition dipole coupling) are very sensitive to tertiary structure, thus the current formalism demonstrates that the Amide I contour may be useful for a more detailed probe of 3-D conformation that goes beyond the traditional use of this band to probe the percentages of particular elements of secondary structure. For example, postulated changes to a known structure can be tested by comparing the new simulated band to the experimental band. A number of refinements to the transition dipole interaction calculation have been made. Most of the important interactions between the C=O oscillators that define the Amide I mode appear to have been identified, including through space transition dipole coupling, through valence bond and through hydrogen bond coupling. The eigenvector matrix produced by the method permits the contribution of each peptide group to the spectrum to be precisely determined. Analysis of the results shows that the often-used structure-frequency correlations are at best approximate and at worst misleading. The subbands from helices, sheets, turns, and loops are much broader and more overlapped than has been commonly assumed. Furthermore, the traditional alpha-helical marker band may be substantially distorted in short segments. Difference spectra based on isotope editing, a technique thought capable of revealing the spectral contributions of individual peptide groups, are shown to be prone to misinterpretation.     

A sphere-based model for the electrostatics of globular proteins

We propose a model for the electrostatics of globular proteins in which the low dielectric region is replaced by concentric spheres of the appropriate size. The method uses analytical formulas for the dielectric sphere and allows an efficient and accurate treatment of bulk charges. For surface charges, we propose a numerical determination of the sphere radius based on the solvent exposure of the individual atoms. The present implementation of the sphere model yields a good approximation of finite-difference Poisson solvation and interaction energies for a test set of 12 proteins.     

An approach to the structure and spectra of copper barbiturate trihydrate

In the neutral title complex [Cu(C₄N₂H₃)₂(H₂O)₃] or [Cu(BBR)₂(H₂O)₃] (BBR^– = Barbiturate), the Cu^II ion, in the slightly distorted square-pyramidal geometry, is coordinated by two O atoms of the two monodentate barbiturates and three O atoms of three water ligands. The average bond length of Cu—O (BBR^–) is 1.981(5) Å and the average bond length of Cu—O (H₂O) at the basal sites is 1.94(5) Å, i.e. much shorter than that of Cu—O (H₂O) [2.175(11) Å]. The crystal structure is characterized by an extensive network of hydrogen bonds in which each [Cu(BBR)₂(H₂O)₃] entity links to six adjacent [Cu(BBR)₂(H₂O)₃] by O(C=O) ··· H—O(H₂O) bonds. Tautomerism in the coordination process for BBR^– was found from the crystal structure and i.r. spectral analysis. The interaction of Cu^II and BBR^– in aqueous solution was also investigated by electronic spectra and electrochemical method. It was observed that the copper ion could not only form the [Cu(BBR)₂(H₂O)₃] complex in aqueous but also catalyze the decomposition of BBR^– at pH 1.1.     

Intermolecular interactions of globular proteins in the crystal state

The present work is devoted to investigation of thermal transitions in the crystals of seven proteins to compare the protein globule stability in crystal and solution. Calorimetry methods, electron and optical microscopy, as well as x-ray diffraction studies are used. It is found that protein crystals do not melt and that the destruction of the crystal lattice is a result of protein globule denaturation within the crystal. It is demonstrated that during the heating of pepsin and DF-trypsin crystals it is possible to observe phase transition of the first order. Equilibrium temperatures of protein denaturation in crystals and in solution coincide. The peculiarities of the crystal state are revealed in the increasing thermal transition cooperativity and the system relaxation period.     

On the zopiclone enantioselective binding to human albumin and plasma proteins. An electrokinetic chromatography approach

In this work, a methodology for the chiral separation of zopiclone (ZPC) by electrokinetic chromatography (EKC) using carboxymethylated-β-cyclodextrin as chiral selector has been developed and applied to the evaluation of the enantioselective binding of ZPC enantiomers to HSA and total plasma proteins. Two mathematical approaches were used to estimate protein binding (PB), affinity constants (K(1)) and enantioselectivity (ES) for both enantiomers of ZPC. Contradictory results in the literature, mainly related to plasma protein binding reported data, suggest that this is an unresolved matter and that more information is needed. Discrepancies and coincidences with previous data are highlighted.     

Contribution of charged groups to the enthalpic stabilization of the folded states of globular proteins

In this paper we use the results from all-atom molecular dynamics (MD) simulations of proteins and peptides to assess the individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the "unfolded state") and charged amino acids in globular proteins (the "folded state"). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO(-)) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while N(+)(3) groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously.     

On the molecular origin of cold denaturation of globular proteins

A polypeptide chain can adopt very different conformations, a fundamental distinguishing feature of which is the water accessible surface area, WASA, that is a measure of the layer around the polypeptide chain where the center of water molecules cannot physically enter, generating a solvent-excluded volume effect. The large WASA decrease associated with the folding of a globular protein leads to a large decrease in the solvent-excluded volume, and so to a large increase in the configurational/translational freedom of water molecules. The latter is a quantity that depends upon temperature. Simple calculations over the -30 to 150 °C temperature range, where liquid water can exist at 1 atm, show that such a gain decreases significantly on lowering the temperature below 0 °C, paralleling the decrease in liquid water density. There will be a temperature where the destabilizing contribution of the polypeptide chain conformational entropy exactly matches the stabilizing contribution of the water configurational/translational entropy, leading to cold denaturation.     

Binary adsorption of globular proteins on ion-exchange media

Adsorption equilibrium of binary pairs of lysozyme (LYS), cytochrome c (CYC) and ribonuclease A (RNase) has been measured on different cation-exchange media at various solution conditions. Adsorption patterns largely follow the intrinsic protein–surface interactions, but can differ significantly for different pairs or even for one pair at different solution conditions. LYS/CYC adsorption shows similar behavior on all the adsorbents examined, with competitive adsorption dominated by LYS and the presence of LYS reducing the adsorption of CYC significantly. Simultaneous and sequential measurements for LYS/CYC show that the order of adsorption does not have a significant effect on the adsorption equilibrium. For LYS/RNase, LYS is consistently more strongly adsorbed. For CYC/RNase, both proteins can display significant adsorption, depending on the pH and salt concentration. A model based on colloidal energetics is developed to calculate the binary adsorption isotherms using parameter values obtained from single-component isotherms. The calculated adsorption is in good agreement with experimental results, with significantly better representation than for other commonly used binary isotherms.     

Interaction of globular proteins with mixed lipid vesicles

Vesicles of charged (Phosphatidic Acid) and neutral (Phosphatidylcholine) lipids were used as membranes model to examine the lateral phase separation induced by a globular protein, namely lysozyme. The ability of the positively charged protein, adsorbed onto vesicles surfaces, to induce the formation of micro-domains richer in the charged lipid component has been investigated by calorimetric measurements, using a DSC instrument. The protein adsorption, rate of denaturation and lipid micro-domains formation were affected bypH and salt concentration variations showing the deep influence of the electric charges in modulating this phenomena.Some of the present results have been rationalized on the basis of a theoretical model recently developed by the authors.     

Role of solvent for globular proteins in solution

The properties of the solvent affect the behavior of the solution. We propose a model that accounts for the contribution of the solvent free energy to the free energy of globular proteins in solution. For the case of an attractive square-well potential, we obtain an exact mapping of the phase diagram of this model without solvent to the model that includes the solute-solvent contribution. In particular we find for appropriate choices of parameters upper critical points, lower critical points, and even closed loops with both upper and lower critical points similar to those found before [Macromolecules 36, 5843 (2003)]. In the general case of systems whose interactions are not attractive square wells, this mapping procedure can be a first approximation to understand the phase diagram in the presence of solvent. We also present simulation results for both the square-well model and a modified Lennard-Jones model.