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Journal Issue - Volume 8 Issue 8 (1999)

Abstract A database of hydrogen‐deuterium exchange results has been compiled for proteins for which there are published rates of out‐exchange in the native state, protection against exchange during folding, and out‐exchange in partially folded forms. The question of whether the slow exchange core is the folding core (Woodward C, 1993, Trends Biochem Sci 18:359–360) is reexamined in a detailed comparison of the specific amide protons (NHs) and...

Abstract We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced...

Abstract The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide‐coupled folding into the cystine‐stabilized α‐helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding...

Abstract Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild‐type and seven site‐specific charge‐reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the...

Abstract The urea‐induced equilibrium unfolding of the α subunit of tryptophan synthase (aTS), a single domain α/β barrel protein, displays a stable intermediate at ˜3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784–792). The same experiment, monitored by one‐dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that...

Abstract Using homology search, structure prediction, and structural characterization methods we show that the C‐terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled‐related proteins, and (4) type I procollagen C‐proteinase enhancer proteins (PCOLCEs) are homologous with the N‐terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR...

Abstract The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine‐scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well‐defined “knobs‐into‐holes” interactions. The bulk of the free energy of binding...

Abstract The structure and dynamics of the fatty acid binding cavity in I‐FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I‐FABP, the probe occupied cavity volume and surface are 539 ± 8 Å3 and 428 Å2, respectively (1.4 Å probe). A total of 31 residues contact the cavity with their side chains. The side‐chain cavity surface is partitioned according to the residue type as follows: 36–39%...

Abstract Lipidation catalyzed by protein prenyltransferases is essential for the biological function of a number of eukaryotic proteins, many of which are involved in signal transduction and vesicular traffic regulation. Sequence similarity searches reveal that the α‐subunit of protein prenyltransferases (PTα) is a member of the tetratricopeptide repeat (TPR) super‐family. This finding makes the three‐dimensional structure of the...

Abstract Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation‐prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners—maltose‐binding protein...

Abstract The phage 434 Cro protein, the N‐terminal domain of its repressor (R1–69) and that of phage λ (λ6–85) constitute a group of small, monomeric, single‐domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in λ6–85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1–69 in 7 M urea has been proposed as a folding initiation site. To...

Abstract Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8‐anilino‐1‐naphthalene sulfonate (ANS). The...

Abstract Heterologous gene expression in either (1) the glycosylation‐defective, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the α‐glucosidase inhibitor, N‐butyldeoxynojirimycin facilitates the trimming of N‐linked glycans of glycoproteins to single N‐acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the ...

Abstract The monomer‐dimer equilibrium for the human immunodeficiency virus type 1 (HIV‐1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in β‐sheet structure and a lower degree of ANS binding. An autolysis‐resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild‐type enzyme is typically unstable in...

Abstract Homologs of the tumor suppressor p53, called p63 and p73, have been identified. The p63 and p73 family members possess a domain structure similar to p53, but contain variable C‐terminal extensions. We find that some of the C‐terminal extensions contain Sterile Alpha Motif (SAM) domains. SAM domains are protein modules that are involved in protein‐protein interactions. Consistent with this role, the C‐terminal SAM domains of...

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