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Journal Issue - Volume 5 Issue 2 (February 1996)

Abstract A general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein...

Abstract The thrombin‐bound structures of native peptide fragments from the fifth EGF‐like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF‐like structure of an antiparallel β‐sheet, a novel structural motif observed for a bound peptide in protein‐peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate...

Abstract Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys‐2...

Abstract We describe a suite of programs, PROMOTIF, that analyzes a protein coordinate file and provides details about the structural motifs in the protein. The program currently analyzes the following structural features: secondary structure; β‐ and γ‐turns; helical geometry and interactions; β‐strands and β‐sheet topology; β‐bulges; β‐hairpins; β‐α‐β units and Ψ‐loops; disulphide bridges; and main‐chain hydrogen bonding patterns. PROMOTIF creates...

Abstract The crystallographic structures of the ternary complexes of human α‐thrombin with hirugen (a sulfated hirudin fragment) and the small‐molecule active site thrombin inhibitors BMS‐186282 and BMS‐189090 have been determined at 2.6 and 2.8 Å. In both cases, the inhibitors, which adopt very‐similar bound conformations, bind in an antiparallel β‐strand arrangement relative to the thrombin main chain in a manner like that...

Abstract MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild‐type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous...

Abstract CD23, a type II membrane receptor protein, recognizes four different ligands via its extracellular C‐type lectin domain: immunoglobulin E (IgE), CD21, and the β2‐integrins CD11b and CD11c. CD23 specifically interacts in a calcium‐dependent manner, “lectin‐like” with carbohydrate moieties expressed on CD21 and CD11b/c, but also “lectin‐unlike” with protein epitopes on IgE. As a first step in analyzing the multiple binding...

Abstract The catalytic rate of wild type, two single (Lys 120 → Leu, Lys 134 → Thr), and one double (Lys 120 → Leu‐Lys 134 → Thr) mutants of Xenopus laevis B Cu, Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the activity of the wild‐type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double...

Abstract We describe a computer algorithm for predicting the three‐dimensional structures of proteins using only their amino acid sequences. The method differs from others in two ways: (1) it uses very few energy parameters, representing hydrophobic and polar interactions, and (2) it uses a new “constraint‐based exhaustive” searching method, which appears to be among the fastest and most complete search methods yet available for...

Abstract The structure and dynamics of the N‐terminal activation domains of the yeast heat shock transcription factors of Kluyveromyces lactis and Saccharomyces cerevisiae were probed by heteronuclear 15N[1H] correlation and 15N[1H] NOE NMR studies. Using the DNA‐binding domain as a structural reference, we show that the protein backbone of the N‐terminal activation domain undergoes rapid, large‐amplitude motions and is therefore unstructured. Difference...

Abstract Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3–5 M), well below that required for complete denaturation, as established by fluorescence and size‐exclusion chromatography. In contrast to the wild‐type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155–188′ and 188–155′, does...

Abstract The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV‐1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV‐1 reverse transcriptase...

Abstract Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged γ‐carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/Ki‐isocitrat/KmNADP) by a factor of 107. Here, we demonstrate that the inactivating effects of the...

Abstract Several members of the ets gene family of transcription factors show negative regulation of DNA binding by intramolecular interactions. A structural mechanism for this auto‐inhibition is investigated using a 161‐residue N‐terminal deletion mutant of murine Ets‐1, Ets‐1ΔN280. This protein shows a similar reduced affinity for DNA as native Ets‐1 because it contains the ETS domain in context of the flanking amino‐ and...

Abstract The equilibrium unfolding reaction of the C‐terminal 80‐amino‐acid dimeric DNA‐binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far‐UV CD, and equilibrium sedimentation. The stability of the HPV‐16 E2 DNA‐binding domain is concentration‐dependent, and the unfolding reaction is well described as a two‐state transition from folded dimer to unfolded monomer. The...

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