Estimation of Distribution Parameters of Organic Solutes in Cloud Point Extraction.

Cloud point extraction of aromatic solutes in the system containing nonionic surfactant polyoxyethylene nonylphenyl ether (PONPE 10) was discussed in terms of their hydrogen-bond donating and accepting abilities and hydrophobicity. It was shown that the Abraham model could be used to estimate the distribution ratio and the effect of aromatic solutes on the cloud point. Copyright 2000 Academic Press.

High-resolution structures of three new trypsin-squash-inhibitor complexes: a detailed comparison with other trypsins and their complexes.

An anionic trypsin from Atlantic salmon and bovine trypsin have been complexed with the squash-seed inhibitors, CMTI-I (Cucurbita maxima trypsin inhibitor I, P1 Arg) and CPTI-II (Cucurbita pepo trypsin inhibitor II, P1 Lys). The crystal structures of three such complexes have been determined to 1.5-1.8 A resolution and refined to crystallographic R factors ranging from 17.6 to 19.3%. The two anionic salmon-trypsin complexes (ST-CPTI and ST-CMTI) and the bovine-trypsin complex (BT-CPTI) have been compared to other trypsin-inhibitor complexes by means of general structure and primary and secondary binding features. In all three new structures, the primary binding residue of the inhibitor binds to trypsin in the classical manner, but with small differences in the primary and secondary binding patterns. Lysine in CPTI-II binds deeper in the specificity pocket of bovine trypsin than lysine in other known lysine-bovine-trypsin complexes, and anionic salmon trypsin lacks some of the secondary binding interactions found in the complexes formed between squash inhibitors and bovine trypsin. The ST-CMTI complex was formed from the reactive-site-cleaved form of the inhibitor. However, well defined electron density was observed for the P1-P1' peptide bond, together with a hydrogen-bonding pattern virtually identical to those of all serine-protease-protein-inhibitor complexes, indicating a resynthesis of the scissile bond.

Protein inhibitors of serine proteinases.

Serine proteinases and their natural protein inhibitors belong to the most intensively studied models of protein-protein recognition. Protein inhibitors do not form a single group but can be divided into about 20 different families. Global structures of proteins representing different inhibitor families are completely different and comprise alpha-helical proteins, beta-sheet proteins, alpha/beta-proteins and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished: canonical (standard mechanism) inhibitors, non-canonical inhibitors, and serpins. The canonical inhibitor binds to the enzyme through the exposed and convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate. Non-canonical inhibitors, originating from blood sucking organisms, specifically block enzymes of the blood clotting cascade. The interaction is mediated through inhibitor N-terminus which binds to the proteinase forming a parallel beta-sheet. There are also extensive secondary interactions which provide an additional buried area and contribute significantly to the strength and specificity of recognition. Serpins are major proteinase inhibitors occurring in plasma. Similarly to canonical inhibitors, serpins interact with their target proteinases in a substrate-like manner. However, in the case of serpins, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.

Variability of the canonical loop conformations in serine proteinases inhibitors and other proteins.

Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in Calpha-Calpha distances as a criterion for loop similarity, we found that the P3-P3' segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (deltar method) and rmsd operating in main chain torsion angles (deltat method), differences of up to 2.1 A and 72.3 degrees, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor-proteinase interaction is very well preserved, as judged from the similarity of Calpha-Calpha distances between Calpha of catalytic Ser and Calpha of P3-P3' residues in various enzyme-inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin-inhibitor complexes are almost always within the range of those for chymotrypsin-inhibitor complexes. Complex formation leads to conformational changes of up to 160 degrees for chi1 angle. Side chains of residue P1 and P2' adopt in different complexes a similar orientation (chi1 angle = -60 degrees and -180 degrees, respectively). To check whether the canonical conformation can be found among non-proteinase-inhibitor Brookhaven Protein Data Bank structures, two selection criteria--the allowed main chain dihedral angles and Calpha-Calpha distances for the P3-P3' segment--were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3-P3' segment and with the scaffold region, P3-P4 and P3'-P4' hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1' residue.

Structural and energetic aspects of protein-protein recognition.

Specific recognition between proteins plays a crucial role in a great number of vital processes. In this review different types of protein-protein complexes are analyzed on the basis of their three-dimensional structures which became available in recent years. The complexes which are analyzed include: those resulting from different types of recognition between proteinase and protein inhibitor (canonical inhibitors of serine proteinases, hirudin, inhibitors of cysteine proteinases, carboxypeptidase inhibitor), barnase-barstar, human growth hormone-receptor and antibody-antigen. It seems obvious that specific and strong protein-protein recognition is achieved in many different ways. To further explore this question, the structural information was analyzed together with kinetic and thermodynamic data available for the respective complexes. It appears that the energy and rates of specific recognition of proteins are influenced by many different factors, including: area of interacting surfaces; complementarity of shapes, charges and hydrogen bonds; water structure at the interface; conformational changes; additivity and cooperativity of individual interactions, steric effects and various (conformational, hydration) entropy changes.