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Journal Issue - Volume 5 Issue 9 (September 1996)

Abstract The amino acid sequence of the flavoprotein subunit of Chromatium vinosum flavocytochrome c—sulftde dehydrogenase (FCSD) was determined by automated Edman degradation and mass spectrometry in conjunction with the three‐dimensional structure determination (Chen Z et al., 1994, Science 266:430–432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass,...

Abstract The crystal structure of the 2[4Fe‐4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 Å resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N‐terminal domain coordinates...

Abstract The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin‐dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein‐protein interactions with its targets. This is not surprising, since p16 is thought to contain...

Abstract Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5′ to 3′ exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N‐terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the StofΔ12 mutant) had no significant effect on activity or stability, but that the further...

Abstract The kinetics of electron transfer from the reduced [2Fe‐2S] ferredoxins from the cyanobacterium Anabaena 7120 and the protozoan Trichomonas vaginalis to select cobalt coordination compounds have been studied in order to gain insight into the mechanism of electron transfer and intrinsic reactivity of [2Fe‐2S] active sites. With tripositive cobalt complexes, reactions of both proteins displayed saturation kinetics; values of association...

Abstract We have devised a Cartesian combination operator and coding scheme for improving the performance of genetic algorithms applied to the protein folding problem. The genetic coding consists of the Cα Cartesian coordinates of the protein chain. The recombination of the genes of the parents is accomplished by: (1) a rigid superposition of one parent chain on the other, to make the relation of Cartesian coordinates meaningful, then, (2) the...

Abstract The optimized g‐tensor parameters for the oxidized form of Rhodobacter capsulatus cytochrome c2 in solution were obtained using a set (50) of backbone amide protons. Dipolar shifts for more than 500 individual protons of R. capsulatus cytochrome c2 have been calculated by using the optimized g‐tensor and the X‐ray crystallographic coordinates of the reduced form of R. capsulatus cytochrome c2. The calculated results for dipolar shifts are compared...

Abstract We present a lattice Monte Carlo study to examine the effect of denaturants on the folding rates of simplified models of proteins. The two‐dimensional model is made from a three‐letter code mimicking the presence of hydrophobic, hydrophilic, and cysteine residues. We show that the rate of folding is maximum when the effective hydrophobic interaction ϵH is approximately equal to the free energy gain ϵS upon forming disulfide bonds. In...

Abstract Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self‐assemble. We describe here a system in which the molecules to be displayed are bound to...

Abstract It is obvious that functional activity of a protein molecule is closely related to its structure. On the other hand, the understanding of structure‐function relationship still remains one of the intriguing problems of molecular biology. There is widespread belief that mutagenesis presents a real way to solve this problem. Following this assumption, we have investigated the effect of circular permutation in dihydrofolate...

Abstract Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide‐linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1–2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their...

Abstract HPLC‐electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1‐R (CFTR residues 404–830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118...

Abstract Human plasma apolipoprotein A‐2 (apoA‐2) is the second major protein of the high‐density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid‐free apoA‐2 in neutral low‐salt solutions is most stable at ∼25 °C and unfolds reversibly both upon heating and cooling from 25 °C. High‐temperature unfolding of...

Abstract Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481–525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most...

Abstract Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4–114 and 4–108, were studied by high‐sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 °C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change δCp = 7.28 ± 0.67 kJ/mol/K, except H56R. The amino...

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