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Journal Issue - Volume 7 Issue 5 (May 1998)

Abstract N52I iso‐2 cytochrome c is a variant of yeast iso‐2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49–56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a “global suppressor” of destabilizing mutations. The equilibrium m‐value for denaturant‐induced unfolding of N52I iso‐2 increases by 30%,...

Abstract Calculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H‐chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three‐fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic...

Abstract The pyridoxal‐P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal‐P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a...

Abstract Tissue factor (TF), a member of the cytokine receptor superfamily, is the obligate cofactor of coagulation factor VIIa (FVIIa), and has a pivotal role in initiating the extrinsic pathway of blood coagulation through formation of the TF‐FVIIa complex. The crystal structure of the extracellular portion of rabbit TF has been solved at 2.35 Å resolution and refined to a crystallographic R‐value of 19.1% (free R‐value, 27.7%)....

Abstract The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq 1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 Å resolution. The dNTPs bind adjacent to the O helix of Klentaq 1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar...

Abstract CD40 Ligand (CD40L) is transiently expressed on the surface of T‐cells and binds to CD40, which is expressed on the surface of B‐cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B‐cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG...

Abstract Two proteins, D‐alanine:D‐alanine ligase and cAMP‐dependent protein kinase, share a remarkable degree of structural convergence despite having different three‐dimensional folds and different enzymatic functions. Here we report that as many as 103 residues from 10 segments form two identical super‐secondary structures between which the cofactor ATP is bound. The cofactor, two bound metal cations, and several water molecules...

Abstract The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D‐lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition...

Abstract A comparison of the three‐dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site‐directed mutagenesis to replace eight complementary ...

Abstract To understand the role of the 23‐amino acid signal sequence in the folding and stability of β‐lactamase, the precursor and a mutantβ‐lactamase with a 19‐amino acid signal sequence deletion were synthesized in vitro using an Escherichia coli cell‐free coupled transcription/translation system. Approximately 30% of the newly synthesized full‐length precursor and 60% of the deletion mutant polypeptides were terminated and released from the...

Abstract Sarcoplasmic reticulum Ca2+‐ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as α‐helix, β‐sheet, etc…, two unusual bands, one at 1,645 cm −1 in H2O buffer and the other at 1,625 cm−1 in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis,...

Abstract Fourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS) was applied for the study of noncovalent chaperone SecB‐ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction ...

Abstract A DnaJ‐like protein, RDJ1, was isolated from a rat brain cDNA library. The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ‐like protein. RDJ1 was also shown to rescue the temperature‐sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1‐151. Fragments containing the J‐domain of RDJl either with or without the G/F motif were expressed in Escherichia coli. The...

Abstract SecB is a chaperone in Escherichia coli dedicated to export of proteins from the cytoplasm to the periplasm and outer membrane. It functions to bind and deliver precursors of exported proteins to the translocation apparatus before they fold into their native structures, thus maintaining them in a competent state for translocation across the membrane. The natural ligands of SecB are precursor proteins containing leader...

Abstract The Z‐score of a protein is defined as the energy separation between the native fold and the average of an ensemble of misfolds in the units of the standard deviation of the ensemble. The Z‐score is often used as a way of testing the knowledge‐based potentials for their ability to recognize the native fold from other alternatives. However, it is not known what range of values the Z‐scores should have if one had a correct...

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