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Journal Issue - Volume 3 Issue 9 (September 1994)

Abstract Transmembrane signal transductions in a variety of cell types that mediate signals as diverse as those carried by neurotransmitters, hormones, and sensory signals share basic biochemical mechanisms that include: (1) an extracellular perturbation (neurotransmitter, hormone, odor, light); (2) specific receptors; (3) coupling proteins, such as G proteins; and (4) effector enzymes or ion channels. Parallel to these...

Abstract Defensins comprise a family of broad‐spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP‐2, one of 4 human...

Abstract Structural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus‐1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer‐dimer‐trimer and monomer‐dimer‐tetramer association models gave the best fit to the analytical ultracentrifuge...

Abstract We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623–8627). The disease arises from substitution of Gly for Asp‐128, a residue at the subunit interface of human aldolase A. To elucidate the role...

Abstract βB2‐ and γB‐crystallins of vertebrate eye lens are 2‐domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of βB2‐ and γB‐domains are very similar, γB‐crystallin is always monomeric, whereas βB2‐crystallin associates to homodimers. It has been suggested that the linker or the protruding N‐ and C‐terminal arms of βB2‐crystallin (not present in γB)...

Abstract We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild‐type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan...

Abstract The fluorescence‐monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 °C, have been quantitatively analyzed using a 3‐state mechanism: US → UF →N. US and UF are slow‐refolding and fast‐refolding unfolded forms of barstar, and N is the native protein. US and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47‐Pro 48 bond. The...

Abstract Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric α‐helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion‐ and pH‐dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid‐denatured proteins, suggesting that...

Abstract We have carried out a series of multiple Xaa → Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed Tm (midpoint unfolding temperature) relative to that predicted from assuming...

Abstract We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 °C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920‐kDa oligomer of 57‐kDa subunits, displays a potassium‐dependent ATPase activity with an optimum temperature at 80 °C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride‐denatured enzymes from...

Abstract The refined structure of dimeric diphtheria toxin (DT) at 2.0 Å resolution, based on 37,727 unique reflections (F > 1σ(F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3′‐5′ uridine 3′ monophosphate (ApUp), and 405 well‐ordered water molecules. The 2.0‐Å refined model reveals that the binding...

Abstract The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 Å resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3′‐5′ uridine 3′ monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical ...

Abstract In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3‐dimensional...

Abstract We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen‐antibody complex followed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI/TOF). A mouse anti‐bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin‐releasing peptide (GRP), was allowed to bind. Direct analysis...

Abstract The substrate‐binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be...

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