Current Protein and Peptide Science - Volume 3, Issue 2, 2002

This article reviews the latest developments in protease-catalyzed peptide synthesis focusing on the use of substrate mimetics. The substrate mimetics approach takes advantage of the characteristic of this novel type of substrates to direct the enzyme to recognize an alternative site on the acyl donor, i.e. the site-specific ester leaving group, mediating the acceptance of originally poorly reactive acyl moieties. At first the kinetics and catalytic mechanism of substrate mimetics-mediated reactions are discussed on the basis of hydrolysis, peptide synthesis, protein-ligand docking, and molecular dynamics studies. By the example of the Glu-specific V8 protease and the aromatic amino acid-specific chymotrypsin both the empirical and computer-aided design of specific substrate mimetics is described. The influence of the leaving group specifically recognized by the enzyme is also considered. The benefits of these artificial substrates over common acyl donor components are illustrated by selected synthesis reactions of small peptides, peptide isosteres, non-peptidic carboxylic acid amides, and the coupling of peptide fragments at non-specific ligation sites resulting in biologically active peptide products. Finally, this review focuses on critical syntheses that uses specific-amino acid-containing peptides as the reactants of ligation. Based on these, the restrictions of the substrate mimetics approach is critically discussed and techniques to their overcoming are presented.

Novel approaches to protein measurement based on mass spectrometry are being developed that challenge more traditional methods. This review summarizes the emergence of mass spectrometry as a tool for clinical protein, peptide, and amino acid determination. Specific applications of mass spectrometry to the measurement of transferrin, transthyretin, glycated hemoglobin, and homocysteine will be discussed, as will the limitations of the technology, and future directions for clinical protein measurement.

The α-helix and β-hairpin are the minimal secondary structure elements of proteins. Identification of the factors governing the formation of these structures independently of the rest of the protein is important for understanding the determinants and rules driving the folding process to a unique native structure. It has been shown that some α-helices and β-hairpins can fold autonomously into native-like structures, either in aqueous solution or in the presence of an organic co-solvent possible mechanisms of these processes have been considered in literature. The characteristic times for folding of α and β structures are estimated from experiments, simple analytical theories and more detailed computer models. Our aim is to review recent experimental and theoretical studies of folding of α and β structures focusing much attention on β-hairpins.

A number of sequence-based analyses have been developed to identify protein segments, which are able to form membrane interactive amphiphilic α-helices. Earlier techniques attempted to detect the characteristic periodicity in hydrophobic amino acid residues shown by these structure and included the Molecular Hydrophobic Potential (MHP), which represents the hydrophobicity of amino acid residues as lines of isopotential around the α-helix and analyses based on Fourier transforms. These latter analyses compare the periodicity of hydrophobic residues in a putative α-helical sequence with that of a test mathematical function to provide a measure of amphiphilicity using either the Amphipathic Index or the Hydrophobic Moment. More recently, the introduction of computational procedures based on techniques such as hydropathy analysis, homology modelling, multiple sequence alignments and neural networks has led to the prediction of transmembrane α-helices with accuracies of the order of 95percent and transmembrane protein topology with accuracies greater than 75percent. Statistical approaches to transmembrane protein modeling such as hidden Markov models have increased these prediction levels to an even higher level. Here, we review a number of these predictive techniques and consider problems associated with their use in the prediction of structure / function relationships, using α-helices from G-coupled protein receptors, penicillin binding proteins, apolipoproteins, peptide hormones, lytic peptides and tilted peptides as examples.

Cell growth at low temperature is dependent on the ability of cells to perform protein synthesis. Cold adapted micro-organisms (psychrophilic or psychrotolerant) have a superior ability to perform translation at low temperature. This review addresses cold adaptation of protein synthesis in Archaea by examining what is presently known about thermal adaptation of elongation factor 2 (EF-2) proteins from Archaea. Despite the knowledge that Archaea are abundant in cold environments (e.g. the ocean), few cold adapted species have been isolated and studied. As a result this review is largely confined to comparative analyses of EF-2 proteins from psychrotolerant (Methanococcoides burtonii) and thermophilic (Methanosarcina thermophila) methanogens. A key finding from these studies is that in addition to inherent properties of the EF-2 proteins, intracellular factors (e.g. ribosomes and intracellular solutes) play a central role in thermal adaptation.

Propeptides of papain-like cysteine proteinases such as papain, cathepsins B, L and S are potent inhibitors of their cognate cysteine proteinases with Ki values in the nanomolar range, and they exhibit highest inhibition selectivity for enzymes from which they originate. Recent studies have identified novel inhibitor proteins that are homologous to the proregions of papain-like cysteine proteinases. Mouse activated T-lymphocytes express cytotoxic T-lymphocyte antigen (CTLA-2), which is homologous to the proregion of mouse cathepsin L. CTLA-2 exhibits inhibitory activities to several cysteine proteinases. We have also identified a similar propeptide-like cysteine proteinase inhibitor, Bombyx cysteine proteinase inhibitor (BCPI), in the silkmoth Bombyx mori. BCPI is a slow and tight binding inhibitor of cathepsin L-like cysteine proteinases with Ki values in picomolar range, and the inhibition is highly selective towards these proteinases just like the propeptides. Recent genome analyses have shown the expression of similar propeptide-like proteins in Drosophila and rat, suggesting the presence of a novel class of cysteine proteinase inhibitors in a variety of organisms. Studies of the gene structures and phylogenetic analysis have shown that genes of the propeptide-like cysteine proteinase inhibitors have emerged from ancestor genes of their parental enzymes.