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Journal Issue - Volume 3 Issue 8 (August 1994)

Abstract The collectins are a group of mammalian lectins containing collagen‐like regions. They include mannan binding protein, bovine conglutinin, lung surfactant protein A, lung surfactant protein D, and a newly discovered bovine protein named collectin‐43. These proteins share a very similar modular domain composition and overall 3‐dimensional structure. They also appear to play similar biological roles in the preimmune defense...

  • The pharmacophore of the human C5a anaphylatoxin

  • Matthew J. Toth, Leslie Huwyler, William C. Boyar, Albert F. Braunwalder, Donna Yarwood, Joseph Hadala, William O. Haston, Matthew A. Sills, Bruce Seligmann, Nicholas Galakatos
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1002/pro.5560030802 (p 1159-1168)

Abstract We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74‐amino acid protein was performed and the effects on the potency of receptor binding and of C5a‐induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein...

Abstract The anaphylatoxin C5a is a pro‐inflammatory factor generated from C5 during complement activation. C5a derived from rat C5 exhibits significantly greater potency compared to C5a from other species. Rat C5a was 25‐fold more potent than human C5a for eliciting spasmogenic contraction of guinea pig ileum. Proteolytic removal of the C‐terminal arginine of C5a (C5adesArg) reduced spasmogenic potency of rat C5a by only 4‐fold compared to a...

Abstract The conversion of substrate, heptenitol, to product, β‐1‐C‐methyl, α‐D‐glucose‐1‐phosphate (heptulose‐2‐P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5‐dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a...

Abstract The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional...

Abstract A recombinant double mutant of hemoglobin (Hb), E6V/L88A(β), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val‐6(β) of one tetramer and the hydrophobic region between Phe‐85(β) and Leu‐88(β) on an adjacent tetramer. Thus, a construct encoding the donor Val‐6(β) of the expressed recombinant HbS and a second mutation encoding an Ala in...

Abstract Site‐directed mutagenesis of an important subunit contact site, Asp‐99(β), by a Lys residue (D99K(β)) was proven by sequencing the entire β‐globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2–15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA,...

Abstract We have analyzed the buried water molecules and internal cavities in a set of 75 high‐resolution, nonhomologous, monomeric protein structures. The number of hydrogen bonds formed between each water molecule and the protein varies from 0 to 4, with 3 being most common. Nearly half of the water molecules are found in pairs or larger clusters. Approximately 90% are shown to be associated with large cavities within the protein,...

Abstract We have examined the pathway and energetics of urea‐induced dissociation and unfolding of the catalytic trimer (C3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N‐(phosphonacetyl)‐L‐Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl− and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the ...

Abstract Reversible unfolding of rat testis fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2‐(iodoacetyl)amino) ethyl)naphthalene‐1‐sulfonic acid) probes. The unfolding reaction is described...

Abstract Absorbance‐detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso‐1‐ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2‐state model. Values for free energy (ΔGD), enthalpy (ΔHD), and entropy (ΔSD) of denaturation were determined as functions of pH and temperature. The value of ΔGD at 300 K, pH 4.6,...

Abstract We have determined the thermodynamic stability and peptide binding affinity of the carboxy‐terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal‐transduction protein Sem‐5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation — at pH 7.3, the protein has a Tm of 73.1 °C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a...

Abstract The 45‐residue C‐terminal EGF‐like domain in human blood coagulation factor IX has been synthesized by a 2‐step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4‐methyl‐benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm...

Abstract The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan‐analogue binding consistently showed positive potential associated with the protein surfaces that...

Abstract The DNA binding domain (DBD) of γΔ resolvase (residues 141–183) is responsible for the interaction of this site‐specific DNA recombinase with consensus site DNA within the γΔ transposable element in Escherichia coli. Based on chemical‐shift comparisons, the proteolytically isolated DBD displays side‐chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu...

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