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Journal Issue - Volume 7 Issue 9 (September 1998)

Abstract The recent sequencing of many complete genomes, combined with the development of methods that allow rapid structure determination for many proteins, has changed the way in which protein structure determinations can be approached. One‐by‐one determinations of individual protein structures will soon be augmented by class‐directed structure analyses in which a group of proteins is targeted and structures of representative...

Abstract The rate constants, kon, for the formation of hen (chicken) lysozyme (HEWL) Fab‐ l0 complexes have been determined for wild‐type (WT) and epitope‐mutated lysozymes by a homogeneous solution method based on the 95% reduced enzymatic activity of the complex. The values fall within a narrow 10‐fold range [(0.18 to 1.92) × 106 M−1 s−1]. The affinity constants, KD, cover a broader, 440‐fold, range from 0.075 to 33 nM. Values of KD as high...

Abstract The hen (chicken) egg‐white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL‐I0 Fab (Fab‐I0) was investigated by alanine scan mutagenesis. The association rate constants (kon) for the HEWL.Fab‐10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (kOff) for the complex, by trapping nascent free antibody...

Abstract The conversion from anα‐helix to a β‐strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self‐assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a β‐strand to anα‐helix by a single‐site mutation as observed in the crystal structure of Fis mutant Pro26 Ala determined at 2.0 Å resolution. Pro26 in Fis occurs at the point where...

Abstract Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure‐based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The...

Abstract Theoretical calculations (Hendsch ZS & Tidor B, 1994, Protein Sci 3:211‐226) and experiments (Waldburger CD et al., 1995, Nut Struct Biol 2:122‐128; Wimley WC et al., 1996, Proc Natl Acud Sci USA 93:2985‐2990) suggest that hydrophobic interactions are more stabilizing than salt bridges in protein folding. The lack of apparent stability benefit for many salt bridges requires an alternative explanation for their occurrence within proteins. To...

Abstract The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arubidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant‐specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arubidopsis. while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely...

Abstract α‐Lactalbumin (α‐LA) undergoes a pH‐dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pigα‐LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual...

Abstract A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [Fe(S‐Cys)4]−1,‐2. The design criteria for the target site included taking backbone movements into account, tetrahedral metal‐binding, and maintaining the structure and stability of the wild‐type...

Abstract The Glu1‐Val79 N‐terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of ˜9 kDa each. K1, K2, K4, and K5 contain each a lysine‐binding site (LBS). Pgn was cleaved with CNBr and the Glu1‐HSer57 N‐terminal fragment (CB‐NTP) isolated. In addition, the Ile27‐Ile56 peptide (L‐NTP) that spans the doubly S‐S bridged loop segment of NTP was synthesized. Pgn kringles...

Abstract Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N‐terminal Glul‐Lys77 peptide (NTP) have been investigated via 1H‐NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM−1). In contrast, while most fragments containing an internal Lys residue exhibit affinities...

Abstract The ubiquitous, multi‐enzyme, nucleotide excision repair (NER) pathway is responsible for correcting a wide range of chemically and structurally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kDa, zinc‐associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and...

Abstract We describe a model for the three‐dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of theα‐family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of...

Abstract The water‐soluble domain of rat hepatic holocytochrome b5 is anαβ protein containing elements of secondary structure in the sequence βl ‐α l ‐β4‐β3‐α2‐α3‐β5‐α4‐α5‐β2‐α6. The heme group is enclosed by four helices,α2,α3,α4, andα5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to βl‐αl‐β4‐β3‐Δ‐β2‐α6 ...

Abstract Amide H/D exchange rates have been measured for the N‐terminal domain of the ribosomal protein L9, residues 1‐56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N‐terminal domain are...

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