Secondary structure prediction of seed storage proteins

The comparison of partial primary structure of seed storage proteins leads to show homologies inside of each considered family (Legume seed legumins and cereal prolamins). Predicted secondary structures deduced from the presently known sequences also exhibit considerable homologies, which implies a severe conservatism of these proteins. Short repetitive segments of sequence of 5-20 residues are frequently occurring and give rise to the prediction either of beta-structure (or alpha-helix) bonded by beta-turns or of successive beta-turns. The latter conformation, which would be able to form a helicoidal arrangement, could contribute to a maximal packing of the protein molecules inside of the subcellular organelles (protein bodies) within which they are confined. As the only known function of seed storage proteins is to provide amino acids to the embryo, it is suggested that their ability to occupy a minimal volume is actually a reasonable explanation of their extreme conservatism in the course of evolution.     

Secondary structure of proteins from circular dichroism spectra. V. Secondary structure of proteins in a "molten globule" state

A method that accounts for the contribution made by aromatic amino acid residues in circular dichroism spectra of proteins has been used in order to analyze the structure of bovine carboanhydrase B, bovine and human alpha-lactalbumin in the native state and when denatured with acid and temperature. At acid- and temperature-induced transitions of the secondary structure of these proteins has been shown not to change. However the rigidity of their tertiary structure decreases (the environment of aromatic amino acid residues is made more symmetrical).     

Secondary structure prediction of human salivary proline-rich proteins

Conformations associated with secondary structure in human salivary proline-rich proteins A (PRPA), C (PRPC), P-D and P-E were predicted by analysis of their respective hydrophobicity profiles by computer programming. Structurally, PRPA and PRPC would present a globular head and a tail that consists of type 3(10) polyproline helices. P-D and P-E would be fibrilar molecules with helical zones of the polyproline 3(10) type. Alternatively for PRPA and PRPC, the head and tail would form one globular domain with the tail folding upon itself at places where random coils occur.     

Secondary structure of proteins and three-dimensional pattern recognition.

We describe a new, parameter-free, method to predict the secondary structure of a protein. It is based on the recognition of well-defined pentapeptides, which allows the discrimination of alpha helices, beta sheets and random coils in proteins. Presently, a success rate of about 65% is achieved for a three-state model.     

Enthalpy changes associated with protein binding to thin films

Molecularly imprinted thin films consisting of proteins embedded in polymerised aminophenyl boronic acid have been made on glass supports. The protein contents of the films have been optimised to achieve a maximum energy of interaction between the film and the native template. The fabrication of the films and the subsequent removal from their surfaces of the imprint proteins has been shown to be a facile and easily reproduced process. The enthalpy changes associated with the rebinding of the films with their original templates (lysozyme and cytochrome c) and with non-native templates has been examined by micro-calorimetry. The results demonstrate that thin films can be successfully imprinted as shown by the significant reduction in the enthalpy (DeltaH) observed when the films were rebound with proteins other than the original templates. Additionally, it was shown that after binding, non-template proteins could be removed by washing and a greater enthalpy again observed when the films were rebound with the native protein compared to that which had been found with the non-native protein.     

Secondary structure of proteins adsorbed onto or embedded in polyelectrolyte multilayers

The structural changes of bovine serum albumin (BSA) and hen egg white lysozyme (HEL) upon their adsorption onto the surface or their embedding into the interior of poly(allylamine hydrochloride)-(poly(styrenesulfonate) (PAH-PSS) multilayer architectures were investigated by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The presence of the polyelectrolytes seems, as previously observed for fibrinogen (J. Phys. Chem. B 2001, 105, 11906-11916), to prevent intermolecular interactions and, thus, protein aggregation at ambient temperature. The secondary structure of the proteins was somewhat altered upon adsorption onto the polyelectrolyte multilayers. The structural changes were larger when the charges of the multilayer outer layer and the protein were opposing. The adsorption of further polyelectrolyte layers onto protein-terminated architectures (i.e., embedding the proteins into a polyelectrolyte multilayer) did not cause considerable further changes in their secondary structures. The capacity of the polyelectrolyte architectures to delay the formation of intermolecular beta-sheets upon increasing temperatures was not uniform for the studied proteins. PSS in contact with HEL could largely prevent the heat-induced aggregation of HEL. In contrast, PAH had hardly any effect on the aggregation of BSA. The differences are explained on the basis of protein-polyelectrolyte interactions, affected mostly by the nature and the strength of the ionic interactions between the polyelectrolyte-protein contact surfaces.     

Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier-transform infrared spectroscopy on hydrated films

Attenuated total reflection Fourier-transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self-deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the alpha-helix and beta-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self-deconvolution/curve-fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/protein ratio can be accurately and quickly determined from the infrared spectrum.     

Effect of calcium concentration on the structure of casein micelles in thin films

The structure of thin casein films prepared with spin-coating is investigated as a function of the calcium concentration. Grazing incidence small-angle x-ray scattering and atomic force microscopy are used to probe the micelle structure. For comparison, the corresponding casein solutions are investigated with dynamic light-scattering experiments. In the thin films with added calcium three types of casein structures, aggregates, micelles, and mini-micelles, are observed in coexistence with atomic force microscopy and grazing incidence small-angle x-ray scattering. With increasing calcium concentration, the size of the aggregates strongly increases, while the size of micelles slightly decreases and the size of the mini-micelles increases. This effect is explained in the framework of the particle-stabilizing properties of the hairy layer of kappa-casein surrounding the casein micelles.     

Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins

Caldesmon binds equally to both gizzard actin and actin containing stoichiometric amounts of bound tropomyosin. The binding of caldesmon to actin inhibits the actin-activation of the Mg-ATPase activity of phosphorylated myosin only when the actin contains bound tropomyosin. The reversal of this inhibition requires Ca2+-calmodulin; but it occurs without complete release of bound caldesmon. Although phosphorylation of the caldesmon occurs during the ATPase assay, a direct correlation between caldesmon phosphorylation and the release of the inhibited actomyosin ATPase is not consistently observed.     

Secondary structure analyses of protein films on gold surfaces by circular dichroism

In order to analyze the secondary structures of protein molecules adsorbed on gold surfaces, circular dichroism (CD) spectra were measured and the secondary structure contents of protein ultra-thin films were estimated quantitatively. A disulfide group was introduced to cytochrome b(562) (cyt.b562), which is a water-soluble b-type heme protein. The cyt.b562 molecules self-assembled to form an ultra-thin protein film both on a gold substrate modified with 2,2(')-dithiodiacetic acid and on a bare gold surface. CD measurements were carried out both in solution and in air, and these results were compared. The protein denaturation was partially prevented, not only in solution but also in air, by both the modification of the substrate and the introduction of the anchor group to the protein molecule. The secondary structure contents of ultra-thin protein films on flat gold surfaces were observed for the first time both in solution and in air by CD spectra.     

Multiple associated proteins regulate proteasome structure and function

We have identified proteins that are abundant in affinity-purified proteasomes, but absent from proteasomes as previously defined because elevated salt concentrations dissociate them during purification. The major components are a deubiquitinating enzyme (Ubp6), a ubiquitin-ligase (Hul5), and an uncharacterized protein (Ecm29). Ecm29 tethers the proteasome core particle to the regulatory particle. Proteasome binding activates Ubp6 300-fold and is mediated by the ubiquitin-like domain of Ubp6, which is required for function in vivo. Ubp6 recognizes the proteasome base and its subunit Rpn1, suggesting that proteasome binding positions Ubp6 proximally to the substrate translocation channel. ubp6Delta mutants exhibit accelerated turnover of ubiquitin, indicating that deubiquitination events catalyzed by Ubp6 prevent translocation of ubiquitin into the proteolytic core particle.     

Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region

Fourier transform spectroscopy in the mid-infrared (400–5,000 cm⁻¹) (FT-IR) is being recognized as a powerful tool for analyzing chemical composition of food, with special concern to molecular architecture of food proteins. Unlike other spectroscopic techniques, it provides high-quality spectra with very small amount of protein, in various environments irrespective of the molecular mass. The fraction of peptide bonds in α-helical, β-pleated sheet, turns and aperiodic conformations can be accurately estimated by analysis of the amide I band (1,600–1,700 cm⁻¹) in the mid-IR region. In addition, FT-IR measurement of secondary structure highlights the mechanism of protein aggregation and stability, making this technique of strategic importance in the food proteomic field. Examples of applications of FT-IR spectroscopy in the study of structural features of food proteins critical of nutritional and technological performance are discussed.     

Secondary structure, mechanical stability, and location of transition state of proteins

It is well known that the unfolding times of proteins, tauu, scales with the external mechanical force f as tauu=tauu0exp(-fxu/kBT), where xu is the location of the average transition state along the reaction coordinate given by the end-to-end distance. Using the off-lattice Go-like models, we have shown that in terms of xu, proteins may be divided into two classes. The first class, which includes beta- and beta/alpha-proteins, has xu approximately 2-5 A whereas the second class of alpha-proteins has xu about three times larger than that of the first class, xu approximately 7-15 A. These results are in good agreement with the experimental data. The secondary structure is found to play the key role in determining the shape of the free energy landscape. Namely, the distance between the native state and the transition state depends on the helix content linearly. It is shown that xu has a strong correlation with mechanical stability of proteins. Defining the unfolding force, fu, from the constant velocity pulling measurements as a measure of the mechanical stability, we predict that xu decays with fu by a power law, xu approximately fu(-mu), where the exponent mu is approximately 0.4. We have demonstrated that the unfolding force correlates with the helix content of a protein. The contact order, which is a measure of fraction of local contacts, was found to strongly correlate with the mechanical stability and the distance between the transition state and native state. Our study reveals that xu and fu might be estimated using either the helicity or the contact order.     

Secondary structure of halorhodopsin

Ultraviolet circular dichroism (CD) spectroscopy in the interval from 190 to 240 nm has been used to estimate the secondary structural content of halorhodopsin (hR), a light-driven chloride pump isolated from the membranes of Halobacterium halobium. Least-squares curve fitting of the CD spectrum for hR solubilized with octyl glucoside yields an alpha-helical content of approximately 50% and a beta-structure content of approximately 30%. The CD spectrum of hR is unaffected by the absence or presence of chloride ions or by the ionic strength of the medium. The CD spectrum of halorhodopsin is very similar to that of bacteriorhodopsin, indicating that these light-driven pumps possess nearly identical fractions of alpha- and beta-secondary structures.     

Secondary structure of histones in solution

The secondary structure of histones H2B and H3 from calf thymus has been quantitatively studied in heavy water solutions in a wide range of histone concentrations, pD, and concentrations of sodium chloride by an infrared spectroscopy method. Also, the interactions between molecules of different histones in equimolar mixtures H2A-H2B, H2A-H3, H2A-H4, H2B-H3, H2B-H4, H3-H4, and H2A-H2B-H3-H4 have been investigated using the same method. For H2B and H3 conditions favourable for aggregation have been shown to induce the formation of pleated sheet structure. When the pD and concentration of NaCl are in a physiological range, the secondary structure of H2B and H3 contains about 15% of alpha-helix, 4% of parallel pleated sheet structure, 14% of antipatallel pleated sheet structure in H2B and 18% in H3. For mixtures in all cases, except H2A-H4, there is an interaction between molecules of different histones followed by a reduction of the antiparallel pleated sheet structure content. The data on the secondary structure of histones in different states (under self-association, in mixtures, in nucleosomes, and in chromatin) have been discussed and it is suggested that: 1) the secondary structure of histones in chromatin is essentially similar to that in the state of self-association; 2) in the core nucleosome particle the quantity of DNA (in nucleotide pairs), and the quantities of alpha-helix and antiparallel pleated sheet structure (in peptide groups) satisfy the relation 1 : 1 : 1.     

Electrical capacitances of thin lipid films

Based on the considerations of surface charge and surface recombination (or ion binding), the capacitances of the diffused double layer and the polar region are calculated as functions of ionic strength, pH value and external bias. Electrical capacitances of thin lipid films were measured under various conditions. In all cases, the change in capacitance is not more than 5%. Agreement between theory and experiment is fairly good except for the capacitance-voltage relation. It is concluded that though the magnitude of the observed membrane capacitance is determined by the center hydrocarbon region, its constancy and stability are attributed to the largeness and guarding action of the interface capacitances.