The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins.

Determinants that govern high-affinity calcium binding.

Genes encoding the minor A component of bovine calbindins D/sub 9k/-the smallest protein known with a pair of EF-hand calcium-binding sites-with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca/sup 2 +/-binding site and constitute Pro-20 ..-->.. Gly (M1), Pro-20 ..-->.. Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13 ..-->.. Phe (M4). /sup 1/H, /sup 43/Ca, and /sup 113/Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca/sup 2 +/-binding site. The Ca/sup 2 +/ exchange rate for the N-terminal site changes from 3 s/sup -1/ in the wild-type protein (M0) and M4 to 5000 s/sup -1/ in M2 and M3, whereas there is no detectable variation in the Ca/sup 2 +/ exchange from the C-terminal site. The macroscopic Ca/sup 2 +/binding constants have been obtained from equilibration in the presence of a fluorescent chelator or by using a Ca/sup 2 +/-selective electrode. Microcalorimetric data show that the enthalpy of Ca/sup 2 +/-binding is negative for all sites except the N-terminal site in M2 and M3.more » The binding entropy is strongly positive in all cases. Through the observed changes in the /sup 1/H NMR spectra during Ca/sup 2 +/-titrations the authors could obtain ratios between site binding constants in M0 and M4. There is evidence (from /sup 113/ Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of ..delta delta..G could not be determined because of sequential Ca/sup 2 +/ binding.« less

Structure-function relationships in EF-hand calcium-binding proteins. Protein engineering and biophysical studies of calbindin D9k

Computational chemical methods have been used to correlate the molecular properties of the 10 ACE inhibitors (captopril, enalapril, perindopril, lisinopril, ramipril, trandolapril, quinapril, fosinopril, benazepril, and cilazapril) and some of their active metabolites (enalaprilat, perindoprilat, ramiprilat, trandolaprilat, quinaprilat, fosinoprilat, benazeprilat, and cilazaprilat). The computed pKa values correlate well with the available experimental values. In the dicarboxylic ACE inhibitors, the carboxyalkyl carboxylate group of the ACE inhibitors studied is more acidic than the C-terminal carboxylate. However, at physiological pH = 7.4 both carboxyl groups of ACE inhibitors are completely ionized and the dicarboxyl-containing ACE inhibitors behave as strong acids. The available experimental partition coefficients of these ACE inhibitors investigated are well reproduced by the neural network-based ALOGPs and the fragment-based KoWWiN methods. All parent drugs (and prodrugs), with the exception of fosinopril, are compounds with low lipophilicity. Calculated pKa, lipophilicity, solubility, absorption, and polar surface area of the most effective ACE inhibitors for the prevention of myocardial infarction, perindopril and ramipril, were found similar. Therefore, it is probable that the experimentally observed differences in the survival benefits in the first year after acute myocardial infarction in patients 65 years of age or older correlate closely to the physicochemical and pharmacokinetic characteristics of the specific ACE inhibitor that is used.

J.D. Buchanan

Papers

The thermodynamics of calcium binding to thermolysin