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Journal Issue - Volume 10 Issue 9 (September 2001)

Abstract The structures of a number of processive enzymes have been determined recently. These proteins remain attached to their polymeric substrates and may perform thousands of rounds of catalysis before dissociating. Based on the degree of enclosure of the substrate, the structures fall into two broad categories. In one group, the substrate is partially enclosed, while in the other class, enclosure is complete. In the latter...

Abstract We have analyzed structure‐sequence relationships in 32 families of flavin adenine dinucleotide (FAD)‐binding proteins, to prepare for genomic‐scale analyses of this family. Four different FAD‐family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p‐cresol methylhydroxylase (PCMH) were previously...

Abstract Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N‐terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild‐type cystatin B and the Cys 3‐to‐Ser mutants of the inhibitors with papain and cathepsins L, H, and...

Abstract Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from α‐thalassemia. α‐Thalassemia is a disease in which there is a deletion of one or more of the four α‐chain genes, and excess γ and β chains spontaneously form homotetramers. The γ4 homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme–heme cooperativity. Although Hb...

Abstract In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild‐type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm....

Abstract Calmodulin (CaM) is a ubiquitous, essential calcium‐binding protein that regulates diverse protein targets in response to physiological calcium fluctuations. Most high‐resolution structures of CaM‐target complexes indicate that the two homologous domains of CaM are equivalent partners in target recognition. However, mutations between calcium‐binding sites I and II in the N‐domain of Paramecium calmodulin (PCaM) selectively...

Abstract Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (ΔR‐SΔH‡) and entropy (ΔR‐SΔS‡) components can be determined. This was done for the resolution of 3‐methyl‐2‐butanol catalyzed by Candida antarctica lipase B and five variants...

Abstract The aggregation of β2‐microglobulin (β2m) into amyloid fibrils occurs in the condition known as dialysis‐related amyloidosis (DRA). The protein has a β‐sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized β2m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single ...

Abstract During evolution of land plants, a specific motif occurred in the N‐terminal domain of the chloroplast‐localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic α‐helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine...

Abstract The designed peptide (denoted 20‐mer, sequence VFITSDPGKTYTEVDPGOKILQ) has been shown to form a three‐strand antiparallel β‐sheet. It is generally believed that the DPro‐Gly segment has the propensity to adopt a type II′ β‐turn, thereby promoting the formation of this β‐sheet. Here, we replaced DPro‐Gly with Asp‐Gly, which should favor a type I′ turn, to examine the influence of different type of turns on the stability of the β‐sheet....

Abstract The sequence and structural analysis of cadherins allow us to find sequence determinants—a few positions in sequences whose residues are characteristic and specific for the structures of a given family. Comparison of the five extracellular domains of classic cadherins showed that they share the same sequence determinants despite only a nonsignificant sequence similarity between the N‐terminal domain and other extracellular...

Abstract Arginine‐140 and isoleucine‐141 were identified as key determinants of 17β‐estradiol (E2) binding affinity of the sex‐steroid‐binding protein (SBP, or SHBG) of human plasma. Amino acid residues that differ between human and rabbit SBP sequences were replaced in the human protein and the products tested for lowered E2binding activity as are seen in the rabbit protein. Only mutants containing either R140K or I141L replacements display an ...

Abstract We showed that the α‐CH2 → NH substitution in octanoyl‐CoA alters the ground and transition state energies for the binding of the CoA ligands to medium‐chain acyl‐CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of...

Abstract The distribution of phosphoglycerate mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor‐dependent and a cofactor‐independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor‐independent PGM, genes homologous to those encoding cofactor‐dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks...

Abstract Because Tyr35β is located at the convergence of the α1β1, α1β2, and α1α2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation‐induced changes in functional and structural properties are found for the recombinant hemoglobins βY35F and βY35A. Oxygen equilibrium‐binding studies in solution, which...

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