Arginyl residues and thermal stability in proteins

Guanidination and amidination of bovine serum albumin, yeast enolase and yeast alcohol dehydrogenase were accompanied by increases in thermal stability at lower extents of modification. Decreases in thermal stability result from greater modification. These results support suggestions that surface guanidino groups (arginyl groups) are an important factor in thermal stability of proteins.     

Amino acid coupling patterns in thermophilic proteins

Structural analysis is useful in elucidating structural features responsible for enhanced thermal stability of proteins. However, due to the rapid increase of sequenced genomic data, there are far more protein sequences than the corresponding three-dimensional (3D) structures. The usual sequence-based amino acid composition analysis provides useful but simplified clues about the amino acid types related to thermal stability of proteins. In this work, we developed a statistical approach to identify the significant amino acid coupling sequence patterns in thermophilic proteins. The amino acid coupling sequence pattern is defined as any 2 types of amino acids separated by 1 or more amino acids. Using this approach, we construct the rho profiles for the coupling patterns. The rho value gives a measure of the relative occurrence of a coupling pattern in thermophiles compared with mesophiles. We found that thermophiles and mesophiles exhibit significant bias in their amino acid coupling patterns. We showed that such bias is mainly due to temperature adaptation instead of species or GC content variations. Though no single outstanding coupling pattern can adequately account for protein thermostability, we can use a group of amino acid coupling patterns having strong statistical significance (p values < 10(-7)) to distinguish between thermophilic and mesophilic proteins. We found a good correlation between the optimal growth temperatures of the genomes and the occurrences of the coupling patterns (the correlation coefficient is 0.89). Furthermore, we can separate the thermophilic proteins from their mesophilic orthologs using the amino acid coupling patterns. These results may be useful in the study of the enhanced stability of proteins from thermophiles-especially when structural information is scarce. Proteins 2005. (c) 2005 Wiley-Liss, Inc.     

Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction

Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.     

Capillary electrophoresis of recombinant proteins

Many naturally occurring proteins which are used therapeutically have been cloned and expressed in large quantities in bacterial, yeast or mammalian systems. Purification of these proteins by column chromatography generates high purity products with low levels of host protein contaminants. However, isoforms of the desired protein may be present at variable concentrations. Analysis of these variant forms has been enhanced by the utilisation of capillary electrophoresis (CE), a highly efficient, widely applicable technique which is increasingly used in the field of biotechnology. The role of CE in the analysis of recombinant proteins is reviewed with respect to microcharacterisation, comparison of natural and recombinant proteins, separation of mutant or variant forms and analysis of glycoforms. Examples of these applications are described and illustrated with analysis of recombinant human albumin. The rapid development of CE, further enhancing its versatility, and its use with complementary analytical techniques is also discussed.     

Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids

Hydrated choline dihydrogen phosphate (Hy[ch][dhp]) containing 30 wt% water was investigated as a novel protein solvent. The Hy[ch][dhp] dissolved some metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and fructose dehydrogenase) without any modification. These proteins retained the surroundings of the active site after dissolution in Hy[ch][dhp]. Some metallo proteins were found to retain their activity in the Hy[ch][dhp]. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1093–1099, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Preparative separation and amino acid composition of neurofilament triplet proteins

Abstract: Intact neurofilaments were isolated from bovine spinal cord white matter, washed by sedimentation in 0.1 m -NaCl, and extracted with 8 m -urea. Solubilized neurofilament triplet proteins of molecular weights approximately 68,000 (P68), 150,000 (P150), and 200,000 (P200) were purified by preparative electrophoresis, using an LKB 7900 Uniphor apparatus. The method provides for an enhanced yield of purified protein and has markedly reduced admixture of electrophoresed protein with acrylamide and associated protein contaminants. Amino acid compositions of the purified neurofilament triplet proteins are reported and compared.     

Influence of thermal history on the stability of gelatin gels

Gelatin gel properties have been studied using three techniques. Optical rotation measurements have shown that the lower the ageing temperature, the faster the helix content increases but the lower the helix stability. Rheological measurements show that a small increase in temperature leads to a melting of some junction zones followed by a build up of new ones. By ageing the gel at two successive temperatures one can show the existence of two populations of junction zones with different thermal stabilities. The same result is shown on melting thermograms obtained by differential scanning calorimetry. All these results are consistent with the hypothesis of the presence of junction zones of various lengths, the thermostability of which being proportional to their lengths.     

Separation used for purification of recombinant proteins

The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.     

Intrinsic contributions of polar amino acid residues toward thermal stability of an ABC-ATPase of mesophilic origin

The nucleotide-binding subunit of phosphate-specific transporter (PstB) from mesophilic bacterium, Mycobacterium tuberculosis, is a unique ATP-binding cassette (ABC) ATPase because of its unusual ability to hydrolyze ATP at high temperature. In an attempt to define the basis of thermostability, we took a theoretical approach and compared amino acid composition of this protein to that of other PstBs from available bacterial genomes. Interestingly, based on the content of polar amino acids, this protein clustered with the thermophiles.     

Amino acid composition of proteins extracted from virions of cytoplasmic-polyhedrosis virus

The ratio of basic to acidic amino acids in protein from free virions of CPV of Malacosoma disstria was 0.26, while that of occluded virions liberated from the polyhedra was 0.43. The ratio in free virions of CPV of Orgyia leucostigma was 0.39. This suggests that the virions of O. leucostigma reach a more advanced stage of maturity before occlusion than do those of M. disstria. By comparison, the composition of the free virions of O. leucostigma CPV was remarkably similar to that of the midgut protein of host cells. On the other hand, the composition of ribosomes of M. disstria was significantly different from either free or occluded virions of M. disstria CPV. Protein amino acids from virions of Bombyx mori differed from those of virions of other hosts in content of basic amino acids yielding a ratio of 0.68. These characteristics help to identify the viral strains and support previous serological studies reported with these proteins.     

Amino acid composition of the proteins from chylomicrons and human serum lipoproteins

By a combination of polyanion precipitation and ultracentrifugation, chylomicrons, very low density, low density, and high density lipoproteins have been isolated from human serum as discrete classes free from contamination with any other major class of lipoprotein or protein. After removal of the lipid, the proteins from each class were hydrolyzed and their amino acid compositions were determined by use of the amino acid analyzer. Application of the "t" test to the concentrations of amino acid residues showed that the amino acid composition of the proteins from each of these lipoprotein classes differs significantly from class to class. However, when the logarithms of the moles of amino acid residues are plotted, there are similarities in the amino acid "profiles" between the chylomicrons and high density lipoproteins on the one hand, and between the very low density and low density lipoproteins on the other. The differences in amino acid composition between the lipoproteins suggest that any metabolic interconversions between them probably do not occur by simple lipolysis.     

Shedding light on the extra thermal stability of thermophilic proteins

An entropic stabilization mechanism has recently gained attention and credibility as the physical ground for the extra thermal stability of globular proteins from thermophilic microorganisms. An empirical result, obtained from the analysis of thermodynamic data for a large set of proteins, strengthens the general reliability of the theoretical approach originally devised to rationalize the occurrence of cold denaturation [Graziano, PCCP 2014, 16, 21755–21767]. It is shown that this theoretical approach can readily account for the entropic stabilization mechanism. On decreasing the conformational entropy gain associated with denaturation, the thermal stability of a model globular protein increases markedly.     

The effects of surface adsorption on the thermal stability of proteins

The effect of surface adsorption on the structure and stability of proteins is a matter of increasing interest in biotechnology. Therefore, we have examined the effect of adsorption to silica on the thermal stability of 7 proteins employing differential scanning calorimetry (DSC) and front surface fluorescence (FSF) spectroscopy. In general, it was found that surface adsorption decreased the thermal stability of the bound protein. Using lysozyme for further studies, DSC, FSF, and FTIR spectroscopies, as well as enzymatic activity measurements, were used to explore the effect of decreasing surface apolarity on stability. It was observed that increasing surface apolarity produced decreasing stability and increasing structural alteration of the adsorbed protein.     

Amino acid composition analysis of secondary transport proteins from Escherichia coli with relation to functional classification,ligand specificity and structure

We have performed an amino acid composition (AAC) analysis of the complete sequences for 235 secondary transport proteins from Escherichia coli, which have functions in the uptake and export of organic and inorganic metabolites, efflux of drugs and in controlling membrane potential. This revealed the trends in content for specific amino acid types and for combinations of amino acids with similar physicochemical properties. In certain proteins or groups of proteins, the so-called spikes of high content for a specific amino acid type or combination of amino acids were identified and confirmed statistically, which in some cases could be directly related to function and ligand specificity. This was prevalent in proteins with a function of multidrug or metal ion efflux. Any tool that can help in identifying bacterial multidrug efflux proteins is important for a better understanding of this mechanism of antibiotic resistance. Phylogenetic analysis based on sequence alignments and comparison of sequences at the N- and C-terminal ends confirmed transporter Family classification. Locations of specific amino acid types in some of the proteins that have crystal structures (EmrE, LacY, AcrB) were also considered to help link amino acid content with protein function. Though there are limitations, this work has demonstrated that a basic analysis of AAC is a useful tool to use in combination with other computational and experimental methods for classifying and investigating function and ligand specificity in a large group of transport or other membrane proteins, including those that are molecular targets for development of new drugs.     

Amino acid composition of the membrane and aqueous domains of integral membrane proteins

To identify residues which might impart transport capability to the intramembranous regions of transport proteins, we surveyed available data for the 9991 amino acids contained in the aqueous and intramembranous regions of 24 integral membrane proteins: 10 transport (T) proteins and 14 nontransport (NT) proteins. Statistical comparison of percentage occurrence of each amino acid within T and NT samples provided a measure of "typical" composition of T and NT membrane-spanning regions, and showed that the residues partition into membrane and aqueous domains largely in accord with expectation from hydropathy indices. Comparison of aqueous and membrane domain composition between protein categories revealed a statistically similar distribution of residues in aqueous domains, but significant differences in membrane domains: seven residues (Asn, Asp, Gln, Glu, Phe, Pro, Tyr) were preferred in membrane regions of T proteins, and one (Val) was selectively excluded. Chemical and structural considerations suggested that three of these residues--Asn, Tyr, and Pro--are the most likely functional participants in transport processes.     

Modification of amino acid composition of endosperm proteins from in-vitro-selected high lysine mutants in rice

Summary Endosperm protein mutants in rice may be recovered by biochemical selections with inhibitory levels of lysine and threonine. Among the phenotypes recovered from in vitro selections are lines with increased protein and percent lysine in the protein. This work was designed to identify changes in proteins of rice mutants and to further our understanding of the mechanisms of lysine plus threonine selections in rice. Among the most obvious amino acid changes in mutants was a higher lysine level in all protein solubility fractions and a decrease in tyrosine. Methionine and glutamate are reduced in some protein fractions. However, methionine is significantly higher in the mutant than the control in the glutelin fraction. Several other aspartate pathway amino acids are higher in the mutant than the unselected controls. Separation of proteins in SDS-PAGE gels showed shifts in the protein profiles in the mutants, including a decrease in the major 30 kDa low lysine globulin component, and an increase in several high-molecular-weight components, approximately 60–100 kDa. Increases in the lysine content of proteins of different solubility classes and different proteins within classes are detailed.