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A protein is usually classified into one of the following four structural classes: all α, all β, (a + B) and α/β. In this paper, based on the maximum correlation-coefficient principle, a new formulation is proposed for predicting the structural class of a protein according to its amino acid composition. Calculations have been made for a development set of proteins from which the amino acid compositions for the standard structural classes were derived, and an independent set of proteins which are outside the development set. The former can test the self consistency of a method and the latter can test its extrapolating effectiveness. In both cases, the results showed that the new method gave a considerably higher rate of correct prediction than any of the previous methods, implying that a significant improvement has been achieved by implementing the maximum-correlation-coefficient principle in the new method. [ABSTRACT FROM AUTHOR]

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]

The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin - NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC 7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (≈ 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved. [ABSTRACT FROM AUTHOR]

The human epidermal-growth-factor receptor (EG F-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor α. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGFR in insect cells using a recombinant baculovirus. From 6 1 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 µM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1 Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20°C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me2+-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGFR. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme. [ABSTRACT FROM AUTHOR]

In this work, the helix-forming residues in fragments of several proteins (ribonuclease, thermolysin, tendamistat and angiogenin) were identified by NOE and the helix proton shifts were measured as δ changes associated with helix-population increments driven by tfifluoroethanol addition. When estimated in this way, a regular pattern of helix conformational shifts was clearly seen in the Δδ versus sequence profiles of ali the peptides studied. The helix periodicity of the Ha and Hβ resonances was especially clear, an observation that earlier statistical studies of protein δ values failed to predict. Amide protons showed the largest helix shifts, but with a less-sharply defined periodic character. Aromatic residues considerably distorted the periodicity of the helix amide shifts in some peptides, as evidenced by the δ shifts of a RNase A fragment 1 - 15 analog in which the two aromatic residues were replaced by Ala. The relationship between helix periodicity and peptide amphiphatic character is discussed. [ABSTRACT FROM AUTHOR]

We have analyzed the amino acid distribution in seven nuclearly encoded and five mitochondrially encoded inner membrane proteins with experimentally well characterized topologies. The mitochondrially encoded proteins conform to the ‘positive inside’ rule, i.e. they have many more positively charged residues in their non-translocated as compared to translocated domains. However, most of the nuclearly encoded proteins do not show such a bias but instead have a surprisingly skewed distribution of Glu residues with an almost ten times higher frequency in the intermembrane space than in the matrix domains. These findings suggest that some, but possibly not all, nuclearly encoded inner membrane proteins may insert into the membrane by a mechanism that does not depend on the distribution of positively charged amino acids. [ABSTRACT FROM AUTHOR]

The S′-subsite specificity of bovine pancreatic α-chymotrypsin was investigated by acyl transfer reactions using a series of amino-acid- and peptide-derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H-Arg-NH2 was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S′ binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water-soluble acyl donor esters, differing both in the P1 residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl-enzyme partitioning between water and added nucleophile in the range of one order of magnitude. [ABSTRACT FROM AUTHOR]

Proton magnetic resonance, circular dichroism and other studies of whole and cleaved calf thymus histone H1 (formerly F1) reveal the presence of specific folded structures in the region approximately from residue 40-115. Ionic, hydrogen-bond and hydrophobic interactions all appear to contribute to the stability of the structure, which is predicted to contain a-helices in regions 42-55 and 58-75. No evidence was found for β-structures, either inter or intramolecular, or for any structure formation outside the region 40-115. At 18 °C and a protein concentration of 2 mM the first-order exchange rate between random-coil and structured forms is slower than 80 s-1; at 40°C the exchange rate is faster than 330 s-1. [ABSTRACT FROM AUTHOR]