Protein-DNA interactions: structural, thermodynamic and clustering patterns of conserved residues in DNA-binding proteins

Amino acid residues, which play important roles in protein function, are often conserved. Here, we analyze thermodynamic and structural data of protein-DNA interactions to explore a relationship between free energy, sequence conservation and structural cooperativity. We observe that the most stabilizing residues or putative hotspots are those which occur as clusters of conserved residues. The higher packing density of the clusters and available experimental thermodynamic data of mutations suggest cooperativity between conserved residues in the clusters. Conserved singlets contribute to the stability of protein-DNA complexes to a lesser extent. We also analyze structural features of conserved residues and their clusters and examine their role in identifying DNA-binding sites. We show that about half of the observed conserved residue clusters are in the interface with the DNA, which could be identified from their amino acid composition; whereas the remaining clusters are at the protein-protein or protein-ligand interface, or embedded in the structural scaffolds. In protein-protein interfaces, conserved residues are highly correlated with experimental residue hotspots, contributing dominantly and often cooperatively to the stability of protein-protein complexes. Overall, the conservation patterns of the stabilizing residues in DNA-binding proteins also highlight the significance of clustering as compared to single residue conservation.     

Studies concerning the specificity of the effect of leucine on the turnover of proteins in muscles of control and diabetic rats.

The protein anabolic effect of branched chain amino acids was studied in isolated quarter diaphragms of rats. Protein synthesis was estimated by measuring tyrosine incorporation into muscle proteins in vitro. Tyrosine release during incubation with cycloheximide served as an index of protein degradation. In muscles from normal rats the addition of 0.5 mM leucine stimulated protein synthesis 36--38% (P less than 0.01), while equimolar isoleucine or valine, singly or in combination were ineffective. The three branched chain amino acids together stimulated no more than leucine alone. The product of leucine transamination, alpha-keto-isocaproate, did not stmino norborane-2-carboxylic acid (a leucine analogue) were ineffective. Leucine and isoleucine stimulated protein synthesis in muscles from diabetic rats.Leucine, isoleucine, valine and the norbornane amino acid but not alpha-ketoisocaproate or beta-hydroxybutyrate decreased the concentration of free tyrosine in tissues during incubation with cycloheximide; tyrosine release into the medium did not decrease significantly. Leucine caused a small decrease in total tyrosine release, (measured as the sum of free tyrosine in tissues and media), suggesting inhibition of protein degradation. The data suggest that leucine may be rate limiting for protein synthesis in muscles. The branched chain amino acids may exert a restraining effect on muscle protein catabolism during prolonged fasting and diabetes.     

Efficacy of antihyperglycemic therapies and the influence of baseline hemoglobin A(1C): a meta-analysis of the liraglutide development program

ObjectiveTo compare Iiraglutide versus common antihyperglycemic treatments in reducing hemoglobin A_1c (A1C) values across multiple levels of baseline glycemic control and in reaching glycemic targets.MethodsPooled patient data from 7 phase 3, multinational, randomized controlled trials in patients with type 2 diabetes were stratified by baseline A1C values into 5 categories: ≤ 7.5%, > 7.5% to 8.0%, > 8.0% to 8.5%, > 8.5% to 9.0%, and > 9.0%. The changes in A1C from baseline to week 26 of treatment and patient proportions reaching A1C targets of < 7.0% and ≤ 6.5% were compared between liraglutide (1.8 mg daily) and sitagliptin, glimepiride, rosiglitazone, exenatide, and insulin glargine across all baseline A1C categories.ResultsIrrespective of treatment, reductions in A1C levels were generally greater in groups with higher baseline A1C values. After 26 weeks of treatment, liraglutide produced the greatest reductions in A1C values across all baseline categories, ranging from 0.7% to 1.8% (baseline A1C categories ≤ 7.5% to > 9.0%, respectively), followed by insulin glargine (0.3% to 1.5%) and then by glimepiride (0.4% to 1.3%). Generally, larger percentages of patients achieved the A1C target of ≤ 6.5% with liraglutide therapy across all baseline categories (from 62% of patients with A1C values ≤ 7.5% to 10% of patients with A1C values > 9.0%) in comparison with other treatments (ranging from 49% to 0% of patients, respectively). Similarly, greater proportions of patients also reached the A1C target of < 7.0% with liraglutide therapy across all baseline categories (from 83% of patients with A1C values ≤ 7.5% to 25% of patients with A1C values > 9.0%) versus comparators (from 74% to 5% of patients, respectively).ConclusionAcross a wide spectrum of baseline A1C categories, liraglutide is an efficacious treatment option for patients with type 2 diabetes. (Endocr Pract. 2011;17: 906-913)     

The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase.

The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxidase were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines. Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the heme c and the hemes a and a3. The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2. Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket. In cytochrome c oxidase, the structural changes in both hemes refer to more subtle perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states. These changes are qualitatively different to those observed for cytochrome c oxidase upon poly-l-lysine binding. The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively. The spectroscopic studies were paralleled by steady-state kinetic measurements of the same protein combinations. The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants. The complex stability decreases in the order: wild-type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine). This order is not exhibited by the conformational equilibria. The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cytochrome c that is defined by the lysyl residues at positions 72, 73 and 79. On the other hand, the conformational changes in cytochrome c oxidase were found to be independent of the identity of the bound cytochrome c variant. The maximum rate constants determined from steady-state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis leads to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1. These results can be interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.     

Studies on cytochrome c oxidase, VII. Isolation and chemical characterization of polypeptide VII.

Polypeptide VII of cytochrome c oxidase was isolated and purified by gel filtration on Bio-Gel P-10 in 10% acetic acid. Automatic Edman degradation of this peptide chain was not successful, because it is blocked at the N-terminus. The amino acid analysis shows a relatively high content of hydrophilic residues (54%). On the basis of this analysis and the apparent molecular weight by sodium dodecyl sulfate gel electrophoresis and gel filtration, a chain length of about 80 residues was calculated. Among the tryptic peptides one blocked heptapeptide was found. Cleavage of this peptide with thermolysin gave two peptide fragments, one of which was not retained on a cation exchange resin. Mass spectrometric sequence determination of this peptide revealed the structure Ac-Ala-Glu-Asp for the N-terminus of polypeptide VII. Treatment with carboxypeptidase A at two different pH values showed that the C-terminal amino acid is isoleucine and the penultimate amino acid is lysine.     

Protein interactions and fluctuations in a proteomic network using an elastic network model

A set of protein conformations are analyzed by normal mode analysis. An elastic network model is used to obtain fluctuation and cooperativity of residues with low amplitude fluctuations across different species. Slow modes that are associated with the function of proteins have common features among different protein structures. We show that the degree of flexibility of the protein is important for proteins to interact with other proteins and as the species gets more complex its proteins become more flexible. In the complex organism, higher cooperativity arises due to protein structure and connectivity.