Protein and Peptide Letters - Volume 12, Issue 5, 2005

Interaction of proteinase inhibitors with proteinases is one of the best understood protein-protein interaction systems. The past decade has seen explosive growth in our understanding of the structure and function of proteinase inhibitors and their molecular basis of interaction with proteinases. It is fitting that this issue of Protein and Peptide Letters carries articles on different aspects of inhibitor structure and functio Read More

Michael Laskowski Jr. (1930-2004) was a pioneer in the field of Standard mechanism serine proteinase inhibitors. He made numerous important contributions in the field. This article highlights some of his most important contributions such as the discovery of the reactive site in serine proteinase inhibitors, the proposal of the Standard mechanism of inhibition, and the sequence to reactivity algorithm for the Kazal famil Read More

We report our progress in understanding the structure-function relationships for the interaction between BPTI and serine proteases. We focused on extensive mutagenesis of four crucial positions from the protease binding loop of BPTI. Selected variants were characterized by determination of association constants, stability parameters and structures of protease-inhibitor complexes.

The members of the Pacifastin family are serine protease inhibitors found in insects and crustacean. They are either small inhibitors (made of one consensus cysteine-rich motif) or proteins (4-9 motifs). Some of these inhibitors are characterized by a species selectivity for the trypsin inhibition. Structural data discriminate the small inhibitors that apparently look very similar into two groups. Interestingly, the inhibitors that display Read More

Potent inhibitors of the Kex2/furin family precursor processing proteases were developed by randomizing adventitious contact sites and screening for optimized affinity using inhibition assays in 96-well format [1]. In this review, the binding interactions of the developed inhibitors will be examined in light of the three dimensional structures of Kex2 and furin [2-4].

Potato type II serine proteinase inhibitors are proteins that consist of multiple sequence repeats, and exhibit a multidomain structure. The structural domains are circular permutations of the repeat sequence, as a result of intramolecular domain swapping. Structural studies give indications for the origins of this folding behaviour, and the evolution of the inhibitor family.

Thrombin is the last enzyme in the blood coagulation cascade. All pharmacological aspects support the use of thrombin inhibitors as antithrombotic agents. Here, we review the unusual inhibition behavior of the highly selective 'reversible suicide substrate' N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-α-azalysine p-nitrophenyl ester (Eoc-D-Phe-ProazaLys- ONp) targeted to the active center of human a-thrombin. Eoc-D-Phe-Pro-az Read More

Evidence that establishes the mechanism of the classes of plant proteinase inhibitors (PIs) is evaluated. Of the eight classes of PIs, six are unique to plants. Except for plant serpins, there is evidence that PIs from all other classes form tight binding complexes with their target proteinases, and that they follow the standard mechanism of inhibition.

The interaction of peptidic inhibitors with serine proteases is investigated using peptide spot synthesis on cellulose sheets. This method permits a highly parallel analysis of subsite specificities and an optimization of peptidic inhibitors towards higher affinity and specificity for a given target protease. Information from crystal structures of corresponding protease/inhibitor complexes may help to explain the observed binding pattern.

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made r Read More

In single domain, “standard mechanism” protein inhibitors of serine proteinases, about a dozen residues make contact with the cognate enzyme. The remainder of the molecule, the scaffolding, holds the reactive site region of the inhibitor in a canonical conformation, improves the binding by about six orders of magnitude and protects it from proteolysis. However, the stability and global structure of the scaffolding is irrelevant Read More

In this paper, a 3D map of protein fold space was produced using Dali structure alignment and nonmetric multidimensional scaling. The fold space comprises four radial clusters, which correspond to the four classes of SCOP. The overall structure of the protein fold space is largely determined by three factors: secondary structure composition, topology of β sheet, and domain size.

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears pr Read More

The local fluorescence probes, 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and NADPH were employed to detect urea-induced conformation changes at each active site of dihydrofolate reductase (DHFR), respectively. The results indicate that local conformation change at DHF/TNS could be superimposed by the conformation change calculated from the enzyme activity change with a three-state model; while at NADPH sit Read More

Human brain serine racemase (hSR) was expressed in large amounts in E. coli with N-terminal His-tag (HishSR). His-hSR expressed in inclusion body was solubilized and purified to homogeneity by Ni-NTA affinity column. Purified His-hSR was refolded in Tween 20/cycloamylose with ∼50% efficiency, and refolded His-hSR was isolated by Q Sepharose column chromatography. The refolding conditions are described in detail. Read More

In this study, we analyze the amino acid pairs in human protein C precursor to determine which amino acid pairs are more susceptible to 71 variants from missense mutant human protein C precursor. The results show 85.92% of 71 variants occur at randomly unpredictable amino acid pairs accounting for 61.96% of amino acid pairs in protein C.

Binding of reduced nicotinamide adenine dinucleotide to yeast alcohol dehydrogenase results in a hypsochromic shift of its absorbance maximum at 340 nm. Application of high hydrostatic pressure to the enzymenucleotide complex returns the absorbance maximum to longer wavelengths. This pressure-dependent bathochromic shift validates one of two assignments on the effects of pressure on the kinetics of the enzymati Read More