Can proteins and crystals self-catalyze methyl rotations?

The chi (C(alpha)-C(beta)) torsional barrier in the dipeptide alanine (N-methyl-l-alanyl-N-methylamide) crystal was investigated using ab initio calculations at various levels of theory, molecular mechanics, and molecular dynamics. For one of the two molecules in the asymmetric unit the calculations suggest that rotation around the chi dihedral angle is catalyzed by the crystal environment, reducing by up to approximately 2kT the torsional barrier in the crystal with respect to that in the gas phase. This catalytic effect is present at both low and room temperature and originates from a van der Waals destabilization of the minima in the methyl dihedral potential coming from the nonbonded environment of the side chain. Screening of a subset of the Protein Data Bank with a pharmacophore model reproducing the crystal environment around this side chain methyl identified a protein containing an alanine residue with an environment similar to that in the crystal. Calculations indicate that this chi torsional barrier is also reduced in the protein at low temperature but not at room temperature. This suggests that environment-catalyzed rotation of methyl groups can occur both in the solid phase and in native biological structures, though this effect might be temperature-dependent. The relevance of this catalytic effect is discussed in terms of its natural occurrence and its possible contribution to the low-frequency vibrational modes of molecules.     

Electrochemistry of membrane proteins and protein–lipid assemblies

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A modified soft docking method for proteins and peptides

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Membrane proteins and proteomics: un amour impossible?

Proteome analysis implies the ability to separate proteins as a first step prior to characterization. Thus, the overall performance of the analysis strongly depends on the performance of the separation tool, usually two-dimensional electrophoresis. This review shows how two-dimensional electrophoresis performs with membrane proteins from bacteria or animal or vegetable cells and tissues, the recent progress in this field, and it examines future prospects in this area.     

Controlled hydrolysis of cheese whey proteins using trypsin and α-chymotrypsin

This study examined the production of protein hydrolysates with controlled composition from cheese whey proteins. Cheese whey was characterized and several hydrolysis experiments were made using whey proteins and purified β-lactoglobulin, assubstrates, and trypsin and α-chymotrypsin, as catalysts, at two tem peratures and several enzyme concentrations. Maximum degrees of hydrolysis obtained experimentally were compared to the theoretical values and peptide compositions were calculated. For trypsin, 100% of yield was achieved; for α-chymotrypsin, hydrolysis seemed to be dependent on the oligopeptide size. The results showed that the two proteases could hydrolyze β-lactoglobulin. Trypsin and α-chymotrypsin were stable at 40°C, but a sharp decrease in the protease activity was observed at 55°C.     

Natural and biotech-derived therapeutic proteins: what is the future?

A myriad of novel proteins and ligands of unknown function will be generated by the Human Genomic Project. Due to differences in post-translational processing, proteins produced by recombinant DNA technology may not possess proper biological activity. One way to find their function is to search for their natural counterparts. Proteins are produced in the tissues, and many of them are secreted into plasma and excreted into urine. There is a virtually "unlimited" array of human proteins in our plasma and urine, many of them in a fully active form. They include small molecules like steroids, peptides, and large glycoproteins like human menopausal gonadotropin. A library of plasma and urinary proteins could be developed to serve as a reference for the novel proteins generated by the functional genomic projects.     

Interfacial behavior of plant proteins — novel sources and extraction methods

Understanding the air-water and oil-water interfacial behavior of plant proteins is crucial for developing stable emulsions and foams in food systems. Plant crops are often processed into protein extracts with high purity, which primarily consist of globulins. These globulins are often unable to form stiff interfacial layers owing to their compact and highly aggregated state and have inferior functionality compared with animal-derived proteins from milk or eggs. Much of the current focus is on modifying these proteins, whereas better interface stabilizing functionality can also be obtained by choosing more targeted protein extraction methods. This review will highlight the benefits and drawbacks of current and novel protein sources and protein extraction methods with respect to interfacial properties.     

Alginate–chitosan micro- and nanoparticles for transmucosal delivery of proteins

The properties of protein-containing micro- and nanoparticles that were produced from alginate and chitosan using the methods of layer-by-layer polyelectrolyte adsorption and ionotropic gelation have been compared. The encapsulation efficiency of proteins (aprotinin, interferon, and human insulin), the size and ζ-potential of the particles, the mucin binding, and the protein release under physiological conditions have been studied. The prospects for the possible mucosal application of the particles are discussed.     

Arginylation and methylation double up to regulate nuclear proteins and nuclear architecture in vivo

Protein arginylation and arginine methylation are two posttranslational modifications of emerging importance that involve Arg residues and their modifications. To test a hypothesis that posttranslationally added arginines can be methylated, we used high-precision mass spectrometry and metabolic labeling to find whether posttranslationally added arginines can serve as methylation sites. We identified?a number of proteins in?vivo, on which posttranslationally added Arg have undergone mono- and dimethylation. This double modification predominantly affects the chromatin-containing nuclear fraction and likely plays an important regulatory role in chromatin-associated proteins. Moreover, inhibition of arginylation and Arg methylation results in?a significant reduction of the nucleus size in cultured cells, suggesting changes in chromatin compaction and nuclear architecture. Our findings suggest?a functional link between protein regulation by arginylation and methylation that affects nuclear structure in?vivo.     

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the chromatographic condition, several denatured proteins can be refolded and separated simultaneously in a single chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative     

Modification of N-terminal α-amino groups of peptides and proteins using ketenes

A method of highly selective N-terminal modification of proteins as well as peptides by an isolated ketene was developed. Modification of a library of unprotected peptides XSKFR (X varies over 20 natural amino acids) by an alkyne-functionalized ketene (1) at room temperature at pH 6.3 resulted in excellent N-terminal selectivity (modified α-amino group/modified ε-amino group = >99:1) for 13 out of the 20 peptides and moderate-to-high N-terminal selectivity (4:1 to 48:1) for 6 of the 7 remaining peptides. Using an alkyne-functionalized N-hydroxysuccinimide (NHS) ester (2) instead of 1, the modification of peptides XSKFR gave internal lysine-modified peptides for 5 out of the 20 peptides and moderate-to-low N-terminal selectivity (5:1 to 1:4) for 13 out of the 20 peptides. Proteins including insulin, lysozyme, RNaseA, and a therapeutic protein BCArg were selectively N-terminally modified at room temperature using ketene 1, in contrast to the formation of significant or major amounts of di-, tri-, or tetra-modified proteins in the modification by NHS ester 2. The 1-modified proteins were further functionalized by a dansyl azide compound through click chemistry without the need for prior treatment.     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

Effect of urea on the structure and functional activity of proteins. Chymotrypsinogen A and α-chymotrypsin

The effect of urea on the structural stability and functional activity of globular proteins,viz., chymotrypsinogen A (ChtG) and -chymotrypsin (Cht), was studied over a wide range of concentrations (0.5–6 rnol L-1), and the existence of two different mechanisms of the action of urea on these proteins was demonstrated. No changes in the spatial structure of ChtG were observed in the concentration range from 0,5 to 3 mol L^–1 (region 1). Differential UV spectroscopy shows tile redistribution of aromatic groups between the inner volume and the outer surface of a protein molecule (protein denaturation) at concentrations >3 mol L^–1 (regionII), In regionI, urea changes the kinetic parameters of enzymatic reactions involving Cht, which is explained on the basis of millimeter spectroscopy data by its action on the structure and nucleophilic reactivity of water.Deceased.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 998–1002, April, 1996.     

‘Apples’ and ‘oranges’: comparing the structural aspects of biomineral- and ice-interaction proteins

The title of this review describes structural comparisons of protein classes whose task is to identify and interact with biological solids (minerals and ice). To date, the following trends have been noted: (1) biomineral-interaction proteins typically adopt unfolded, open conformations, and, where mineral binding motifs have been identified, these sequences exhibit structural trends towards extended, random coil, or other unstable secondary structures; (2) ice-interaction proteins typically adopt folded structures, featuring stable secondary structure preferences (α-helix, β-sheet, β-helix, etc.) and stable, planar ice binding motifs that exploit hydrophobicity and van der Waals’ interactions for ice binding.     

Complexation of radionuclides with natural polyelectrolytes — proteins, polysaccharides and humic substances

Various approaches to the modeling of metal and radionuclide interactions with macromolecular ligands, proteins, polysaccharides and humic substances in particular, their chemical and sorption equilibria, and the techniques used for their investigation are concisely compared. To predict radionuclide mobility in the natural and semi-natural aqueous environment, the estimation of the effective interaction constants, related to specific species of polyelectrolytes which are linked with the absorbancy or absorbancy ratio in their electronic absorption spectra, should probably be preferred and developed as standard. For characterization of the binding sites of specific molecular forms of polyelectrolyte ligands, the Scatchard plot analysis at macroconcentrations of the metal and also, though not so effectively, the two-phase distribution at trace concentrations of radionuclide or metal can be applied.     

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and β-purothionin

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and β-purothionin (β-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and β-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and β-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. β-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 ? thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and β-Pth hydrophobic regions.     

Confinement in nanopores can destabilize α-helix folding proteins and stabilize the β structures

Protein folding in confined media has attracted wide attention over the past decade due to its importance in both in vivo and in vitro applications. Currently, it is generally believed that protein stability increases by decreasing the size of the confining medium, if its interaction with the confining walls is repulsive, and that the maximum folding temperature in confinement occurs for a pore size only slightly larger than the smallest dimension of the folded state of a protein. Protein stability in pore sizes, very close to the size of the folded state, has not however received the attention that it deserves. Using detailed, 0.3-ms-long molecular dynamics simulations, we show that proteins with an α-helix native state can have an optimal folding temperature in pore sizes that do not affect the folded-state structure. In contradiction to the current theoretical explanations, we find that the maximum folding temperature occurs in larger pores for smaller α-helices. In highly confined pores the free energy surface becomes rough, and a new barrier for protein folding may appear close to the unfolded state. In addition, in small nanopores the protein states that contain the β structures are entropically stabilized, in contrast to the bulk. As a consequence, folding rates decrease notably and the free energy surface becomes rougher. The results shed light on many recent experimental observations that cannot be explained by the current theories, and demonstrate the importance of entropic effects on proteins' misfolded states in highly confined environments. They also support the concept of passive effect of chaperonin GroEL on protein folding by preventing it from aggregation in crowded environment of biological cells, and provide deeper clues to the α → β conformational transition, believed to contribute to Alzheimer's and Parkinson's diseases. The strategy of protein and enzyme stabilization in confined media may also have to be revisited in the case of tight confinement. For in silico studies of protein folding in confined media, use of non-Go potentials may be more appropriate.     

Structural and dynamic aspects of electron transfer in proteins — highly organized natural nanostructures

The review briefly outlines theoretical models developed in 1990s to describe electron transfer reactions (ETR) in proteins, as well as different variants of improvements in these models proposed by the present authors to describe ETR in reaction centers (RC) of photosynthetic bacteria with consideration of their molecular dynamics in a wide temperature range. Experimental data on electron transfer from reduced proximal heme c-559 of cytochrome to bacteriochlorophyll dimer radical cation P⁺ in RC from two types of bacteria, viz., native and mutant RC from Rps. viridis and native RC from Rps. sulfoviridis were analyzed within the framework of the models which take into account the quantum and classical (including diffusive) degrees of freedom responsible for reorganization of the protein globule.     

Use of γ-irradiation to produce films from whey,casein and soya proteins: structure and functionals characteristics

γ-irradiation and thermal treatments have been used to produce sterilized cross-linked films. Formulations containing variable concentrations of calcium caseinate and whey proteins (whey protein isolate (WPI) and commercial whey protein concentrate) or mixture of soya protein isolate (SPI) with WPI was investigated on the physico-chemical properties of these films. Results showed that the mechanical properties of cross-linked films improved significantly the puncture strength for all types of films. Size-exclusion chromatography showed for no cross-linked proteins, a molecular mass of around 40 kDa. The soluble fractions of the cross-linked proteins molecular distributions were between 600 and 3800 kDa. γ-irradiation seems to modify to a certain extent the conformation of proteins which will adopt structures more ordered and more stable, as suggested by X-ray diffraction analysis. Microstructure observations showed that the mechanical characteristics of these films are closely related to their microscopic structure. Water vapor permeability of films based on SPI was also significantly decreased when irradiated. Microbial resistance was also evaluated for cross-linked films. Results showed that the level of biodegradation of cross-linked films was 36% after 60 d of fermentation in the presence of Pseudomonas aeruginosa.