Analysis of fractal properties of globular protein surfaces by means of small-angle x-ray scattering]

The "cube method" [M. Yu. Pavlov, B. A. Fedorov, Biopolymers, 22, 1507, 1983] has been used to calculate the intensity of the large-angle X-ray scattering from the volumes of several globular proteins. In the logarithmic plots of the scattered intensity curves from three of these proteins, there is a linear region at scattering angles corresponding to Bragg distances of from about 6.3 A to 21 A. This linear region possibly may be due to the fractal properties of the surfaces of these proteins on length scoles from 6.3 A to 21 A, and the fractal dimensions corresponding to the power-law scattering at these scattering angles have been evaluated.     

Diffuse x-ray wide-angle scattering of polyglutamic acid in solution]

The diffuse wide angle x-ray scattering (WAXS) of polyglutamic acid (PGA) in solution was studied using an x-ray diffractometer with small aperture of the primary beam. The scattering curve was recorded at an angular interval from (article: see text). The experimental scattering intensity of PGA with alpha-helical CD spectrum showed a maximum at 14.4 nm-1. Unordered PGA in solution yielded no maximum at this scattering angle. The studies have proved that the scattering theory can be applied to globular proteins in solution as well as to chain molecules in solution in this angular interval. The differences between the calculated scattering curves and the experimental curves indicate minor movements of the side chains of PGA in solutions and slight structuring of the solvent at the surface of the polypeptide chain.     

Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins.

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure beta-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:669-679, 1991), was improved by the addition of proteins with high beta-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (alpha-helix, antiparallel beta-pleated sheet, beta-turns, and unordered conformation), as well as a curve attributed to the "aromatic contribution" in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the alpha-helical structure, but upon analysis yielded a considerable amount of beta-sheet in agreement with the X-ray structure.     

Improved technique for calculating X-ray scattering intensity of biopolymers in solution: Evaluation of the form,volume, and surface of a particle

An improved cube method has been developed for calculating the intensity of diffuse x-ray scattering of macromolecules in solution using a certain set of their atomic coordinates. The technique is based on the ideas of B. Lee and F. M. Richards [(1971) J. Mol. Biol. 55 , 374–400] and Richards [(1977) Annu. Rev. Biophys. Bioeng. 6 , 151–176] on the possibility of estimating the molecular and accessible surface of a particle by “rolling” a sphere, simulating a water molecule, on its molecular surface. It is shown that this technique is more advantageous than earlier versions of the cube methods. The improved technique for calculating scattering curves was utilized for several globular proteins, and for the first time, reliable scattering curves were obtained for protein-“bound” water complexes. In the case of globular proteins and tRNA, this technique has permitted a strict evaluation of their accessible surfaces, their volumes, and, apparently for the first time, their complete molecular surfaces.     

Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.     

Analysis of interactions of buried polar side chains in beta-proteins

It was shown in qualitative and quantitative analyses of polar side chains inaccessible for water molecules as well as their interactions in 100 globular beta-structural proteins that completely buried polar side chains are widespread in beta-proteins, their vast majority being involved in "side chain-side chain" or "side chain-main chain" interactions. The analysis of the occurrence of different "side chain-partner" pairs permitted us to demonstrate that such interactions are selective. The results were compared with similar data obtained previously for alpha-helical proteins.     

Thermodynamic characterization of an artificially designed amphiphilic alpha-helical peptide containing periodic prolines: observations of high thermal stability and cold denaturation.

To investigate the structural stability of proteins, we analyzed the thermodynamics of an artificially designed 30-residue peptide. The designed peptide, NH2-EELLPLAEALAPLLEALLPLAEALAPLLKK-COOH (PERI COIL-1), with prolines at i + 7 positions, forms a pentameric alpha-helical structure in aqueous solution. The thermal denaturation curves of the CD at 222 nm (pH 7.5) show an unusual cold denaturation occurring well above 0 degrees C and no thermal denaturation is observable under 90 degrees C. This conformational change is reversible and depends on peptide concentration. A 2-state model between the monomeric denatured state (5D) and the pentameric helical state (H5) was sufficient to analyze 5 thermal denaturation curves of PERI COIL-1 with concentrations between 23 and 286 microM. The analysis was carried out by a nonlinear least-squares method using 3 fitting parameters: the midpoint temperature, Tm, the enthalpy change, delta H(Tm), and the heat capacity change, delta Cp. The association number (n = 5) was determined by sedimentation equilibrium and was not used as a fitting parameter. The heat capacity change suggests that the hydrophobic residues are buried in the helical state and exposed in the denatured one, as it occurs normally for natural globular proteins. On the other hand, the enthalpy and the entropy changes have values close to those found for coiled-coils and are quite distinct from typical values reported for natural globular proteins. In particular, the enthalpy change extrapolated at 110 degrees C is about 3 kJ/mol per amino acid residue, i.e., half of the value found for globular proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cotranslational, cosecretory protein folding and its renaturation from a denatured state

On the basis of the available experimental data on structure, biosynthesis and secretion of globular proteins it is concluded that an alpha-helix is a starting conformation at formation of the native structure of any globular protein (alpha-helical model for initiation of protein folding). The structural invariant (clusterization of hydrophobic side chains on the alpha-helix surface) in the amino acid sequences of globular proteins is found which is predicted by alpha-helical model for the initiation of protein folding. The model predicts the pyramidization of the atoms C and N of peptide groups during the formation of spatial structure of proteins and a number of other effects that can be put to the experimental test. In the work the mechanism for protein translocation across membrane lipid bilayer is also suggested.     

Low-frequency modes of peptides and globular proteins in solution observed by ultrafast OHD-RIKES spectroscopy

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.     

Effect of the fluctuations in electron density within a globular protein on the excess small-angle X-ray scattering

Excess small angle X-ray scattering in solvents of differing electron density has been calculated from the crystal structures obtained for rubredoxin, trypsin inhibitor, myogen, ferricytochrome c₂, ribonuclease S, lysozyme, nuclease, myoglobin, α-chymotrypsin, elastase, subtilisin, carboxypeptidase A, thermolysin, methemoglobin, deoxyhemoglobin, and a single polypeptide chain of M₄ lactate dehydrogenase. The scattering curves for each protein can be reproduced by the sum of three curves, with the weighting of the three curves depending on the electron density of the solvent. The radius of gyration obtained from the small angle X-ray scattering by globular proteins in aqueous solution will usually exceed the values defined by the shape of the macromolecule. Deviations for certain of the proteins cited are calculated to be as large as 6%. These deviations arise from the tendency for the amino acid residues with low electron density to be situated closer to the center of the protein than the amino acid residues of high electron density. An upper limit of 19% is obtained for the discrepancy between the radius of gyration defined by the shape of a spherical globular protein of typical amino acid composition and the apparent radius of gyration measured for that protein in water by small angle X-ray scattering.     

A simple statistical method for discriminating outer membrane proteins with better accuracy

MOTIVATION: Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for identifying outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS: We have systematically analyzed the amino acid composition of globular proteins from different structural classes and outer membrane proteins. We found that the residues, Glu, His, Ile, Cys, Gln, Asn and Ser, show a significant difference between globular and outer membrane proteins. Based on this information, we have devised a statistical method for discriminating outer membrane proteins from other globular and membrane proteins. Our approach correctly picked up the outer membrane proteins with an accuracy of 89% for the training set of 337 proteins. On the other hand, our method has correctly excluded the globular proteins at an accuracy of 79% in a non-redundant dataset of 674 proteins. Furthermore, the present method is able to correctly exclude alpha-helical membrane proteins up to an accuracy of 80%. These accuracy levels are comparable to other methods in the literature, and this is a simple method, which could be used for dissecting outer membrane proteins from genomic sequences. The influence of protein size, structural class and specific residues for discrimination is discussed.     

Discrimination of outer membrane proteins using support vector machines

MOTIVATION: Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for dissecting outer membrane proteins (OMPs) from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS: We have developed a method based on support vector machines using amino acid composition and residue pair information. Our approach with amino acid composition has correctly predicted the OMPs with a cross-validated accuracy of 94% in a set of 208 proteins. Further, this method has successfully excluded 633 of 673 globular proteins and 191 of 206 alpha-helical membrane proteins. We obtained an overall accuracy of 92% for correctly picking up the OMPs from a dataset of 1087 proteins belonging to all different types of globular and membrane proteins. Furthermore, residue pair information improved the accuracy from 92 to 94%. This accuracy of discriminating OMPs is higher than that of other methods in the literature, which could be used for dissecting OMPs from genomic sequences. AVAILABILITY: Discrimination results are available at http://tmbeta-svm.cbrc.jp.     

Novel approach for alpha-helical topology prediction in globular proteins: generation of interhelical restraints

The protein folding problem represents one of the most challenging problems in computational biology. Distance constraints and topology predictions can be highly useful for the folding problem in reducing the conformational space that must be searched by deterministic algorithms to find a protein structure of minimum conformational energy. We present a novel optimization framework for predicting topological contacts and generating interhelical distance restraints between hydrophobic residues in alpha-helical globular proteins. It should be emphasized that since the model does not make assumptions about the form of the helices, it is applicable to all alpha-helical proteins, including helices with kinks and irregular helices. This model aims at enhancing the ASTRO-FOLD protein folding approach of Klepeis and Floudas (Journal of Computational Chemistry 2003;24:191-208), which finds the structure of global minimum conformational energy via a constrained nonlinear optimization problem. The proposed topology prediction model was evaluated on 26 alpha-helical proteins ranging from 2 to 8 helices and 35 to 159 residues, and the best identified average interhelical distances corresponding to the predicted contacts fell below 11 A in all 26 of these systems. Given the positive results of applying the model to several protein systems, the importance of interhelical hydrophobic-to-hydrophobic contacts in determining the folding of alpha-helical globular proteins is highlighted.     

Prediction of "hot spots" of aggregation in disease-linked polypeptides

Background  

The polypeptides involved in amyloidogenesis may be globular proteins with a defined 3D-structure or natively unfolded proteins. The first class includes polypeptides such as β2-microglobulin, lysozyme, transthyretin or the prion protein, whereas β-amyloid peptide, amylin or α-synuclein all belong to the second class. Recent studies suggest that specific regions in the proteins act as "hot spots" driving aggregation. This should be especially relevant for natively unfolded proteins or unfolded states of globular proteins as they lack significant secondary and tertiary structure and specific intra-chain interactions that can mask these aggregation-prone regions. Prediction of such sequence stretches is important since they are potential therapeutic targets.     

Maps of preferential conformation of dipeptides in the structural regions of globular proteins

According to X-ray crystallographic analysis of 46 globular proteins the probability of dipeptides frequency in alpha-helical beta-sheet and random coil conformations has been studied on the basis of which the maps of preferentially conformational state of dipeptide in different elements of secondary structure of proteins have been obtained.     

Orientation of skeletal muscle actin in strong magnetic fields

Measurement of birefringence is used to follow actin filament and paracrystal formation in a strong magnetic field. Both F-actin and paracrystals orientate parallel to the field. This confirms that globular proteins arranged in filamentous assemblies can orientate in magnetic fields. This is consistent with the alpha-helical component of the actin subunits being approximately aligned along the actin filament.     

Conformation of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> ribosomal protein S1 in solution at different ionic strengths

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 Å, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D _20,w = (2.7 ± 0.1)·10^?7 versus (5.8–6.0)·10^?7 cm² s^?1), confirming protein association under equilibrium conditions.     

Possibility of wide-angle neutron scattering in the study of proteins and nucleic acids in solutions

A method is proposed for calculating wide-angle neutron scattering curves of biopolymers at any fraction of heavy water (D2O) in solution. The method permits to accurately take into account the phenomenon of deuteroexchange. By this method neutron scattering curves of proteins and DNA have been calculated. The calculations have shown that at optimal fractions of D2O in solution the profiles of neutron scattering curves and their sensitivity to conformational rearrangements in protein molecules turned out to differ very little from those of corresponding X-ray curves. Thus the neutron scattering curves do not contain any additional information (as compared with those contained in X-ray scattering curves) on the structure of proteins in solution. On the contrary, neutron and X-ray scattering curves of DNA differ significantly at all fractions of D2O in solution and therefore the methods of wide-angle neutron and X-ray scattering could become mutually complementary in studying the structure of nucleic acids in solution.     

Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.     

Interaction of adjacent amino acid residues in the structured regions of globular proteins

Distribution of the pairs of amino acids i, i + 1 in alpha-helical, beta-sheet and random coil regions from 46 globular proteins comprising 8115 amino acid residues was analyzed. Statistical analysis of the data excludes null hypothesis about random pairing of the amino acid residues i, i + 1 in beta-sheet and random coil configurations. The distribution of the amino acid pairs, i, i + 1 in alpha-helical configurations does not differ from the random pairing.