Biomedical subjects

H A Koteiche

Publications and source records attributed to H A Koteiche.

5 recordsLinked to original sources

PubMed1999

Folding pattern of the alpha-crystallin domain in alphaA-crystallin determined by site-directed spin labeling.

The folding pattern of the alpha-crystallin domain, a conserved protein module encoding the molecular determinants of structure and function in the small heat-shock protein superfamily, was determined in the context of the lens protein alphaA-crystallin by systematic application of site-directed spin labeling. The sequence-specific secondary structure was assigned primarily from nitroxide scanning experiments in which the solvent accessibility and mobility of a nitroxide probe were measured as a function of residue number. Seven beta-strands were identified and their orientation relative to the aqueous solvent determined, thus defining the residues lining the hydrophobic core. The pairwise packing of adjacent strands in the primary structure was deduced from patterns of proximities in nitroxide pairs with one member on the exposed surface of each strand. In addition to identifying supersecondary structures, these proximities revealed that the seven strands are arranged in two beta-sheets. The overall packing of the two sheets was determined by application of the general rules of protein structure and from proximities in nitroxide pairs designed to distinguish between known all beta-sheet folds. Our data are consistent with an immunoglobulin-like fold consisting of two aligned beta-sheets. Comparison of this folding pattern to that of the evolutionary distant alpha-crystallin domain in Methanococcus jannaschii heat-shock protein 16.5 reveals a conserved core structure with the differences sequestered at one edge of the beta-sandwich. A beta-strand deletion in alphaA-crystallin disrupts a subunit interface and allows for a different dimerization motif. Putative substrate binding regions appear to include a buried loop and a buried turn, suggesting that the chaperone function involves a disassembly of the oligomer.

Amino Acid Motifs↗

PubMed1998

Identification of protein folding patterns using site-directed spin labeling. Structural characterization of a beta-sheet and putative substrate binding regions in the conserved domain of alpha A-crystallin.

The folding pattern of the segment of alphaA-crystallin encoded by exon 2 and containing putative substrate binding sites was explored using site-directed spin labeling (SDSL). For this purpose, a nitroxide scan was carried out between residues 60 and 108. At each site, structural constraints describing the local environment and topography were obtained from analysis of the nitroxide mobility and its solvent accessibility. Periodic patterns in the sequence-specific variation of these parameters were used to assign the secondary structure along the sequence. Geometric constraints describing the packing of secondary structure were deduced from patterns of proximities in 20 nitroxide pairs, specifically designed to differentiate between supersecondary structural motifs. Our data, in conjunction with those of Berengian et al. [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951-9957], reveal that the fold of the segment between residues 84 and 120 consists of an antiparallel beta-sheet of three strands arranged in consecutive beta-hairpins. The boundaries of the sheet are defined at one end by a surface of isologous association and on the other end by an unstructured, charged interdomain segment. One of the putative substrate binding segments overlaps a buried loop, suggesting that the structural origin of the thermal activation of binding is the transient exposure of this site. This paper describes and implements a general strategy for experimental fold recognition using SDSL. The results of its application to alphaA-crystallin provide the first experimental insight into the folding pattern of the subunit and establish the structural context necessary to understand molecular recognition and substrate binding.

Amino Acid Sequence↗

PubMed1997

Site-directed spin-labeling study of the structure and subunit interactions along a conserved sequence in the alpha-crystallin domain of heat-shock protein 27. Evidence of a conserved subunit interface.

Site-directed spin-labeling (SDSL) was used to investigate the secondary structure, solvent accessibility, and tertiary and quaternary interactions along the sequence located between residues 133 and 144 in the alpha-crystallin domain of human heat-shock protein 27 (HSP 27). The sequence is conserved among mammalian sHSP and shows similarity to the region of highest homology between alpha A- and alpha B-crystallins. Eleven sequential single cysteine mutants were prepared and reacted with a sulfhydryl-specific spin-label. The accessibilities of attached nitroxide side chains to a paramagnetic probe in the aqueous solution were determined. Spectral line shapes were analyzed in terms of side-chain mobility and spatial proximity to nearby nitroxides. The sequence-specific mobilities and accessibilities varied with a period of 2, consistent with the presence of a beta-strand along the sequence. At even sites, the nitroxide environment is highly ordered with virtually no accessibility to the hydrophilic probe, indicating that one face of the strand is buried. Furthermore, spin-spin interactions between nitroxides in the oligomeric structure strongly suggest that equivalent strands from different subunits are in close spatial proximity. These structural characteristics are remarkably similar to those of the equivalent sequence in alpha A-crystallin [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951-9957]. In both proteins, a beta-strand spans the sequence and is located at a subunit interface, indicating that one set of interactions between subunits, and its associated symmetry, is conserved. This is the first report of sequence-specific structural similarity between alpha A-crystallin and HSP 27 and the first identification of a conserved secondary structural element in the alpha-crystallin domain.

Amino Acid Sequence↗

PubMed1996

Rhodobacter sphaeroides phosphoribulokinase: binary and ternary complexes with nucleotide substrate analogs and effectors.

Rhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP and ATP gamma S-acetamidoproxyl), to form stable binary complexes. Stoichiometric binding of these nucleotide triphosphates in PRK's substrate site is observed not only with wild-type enzyme but also with D42A and D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their substantial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al. (1994) Biochemistry 33, 9343-9350]. Similarly, PRK forms a stable binary complex with the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binary complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleotide triphosphate that functions as an alternative PRK substrate, forms a binary complex with enzyme, its fluorescence emission is enhanced and lambda max shifted from approximately 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the lambda max shifted from approximately 460 to 440 nm. PRK forms stable ternary complexes containing NADH and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH fluorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between this novel donor/acceptor pair.

Adenosine Triphosphate↗

PubMed1995

Utility of a novel spin-labeled nucleotide in investigation of the substrate and effector sites of phosphoribulokinase.

The activated spin-label 3-(2-bromoacetamido)proxyl modifies the sulfur atom of phosphorothioate-containing AMP, ADP, and ATP analogs in a facile reaction that produces a new series of spin-labeled nucleotides. One of these products, adenosine 5'-O-(S-acetamidoproxyl 3-thiotriphosphate) (ATP gamma SAP), has been evaluated as a structural probe for Rhodobacter sphaeroides phosphoribulokinase (PRK). When incubated with affinity-purified enzyme that contains tightly bound substrate ATP, ATP gamma SAP binds noncooperatively to the allosteric site (n = 1; KD = 8 microM). Probe bound in this site is displaced (K1/2 = 100 microM) by the allosteric effector, NADH, at concentrations comparable to those required for enzyme activation (Ka = 133 microM). In the presence of NADH, when PRK's substrate site is vacant, ATP gamma SAP binds in a cooperative mode (Hill coefficient approximately 2.9; KD = 20 microM). In the absence of NADH, ATP gamma SAP mimics ATP by exhibiting nonequilibrium binding to PRK. The observations with phosphoribulokinase, together with the straightforward nature of the methodology documented for synthesis and isolation of this class of spin-labeled nucleotides, suggest that these analogs have potentially wide application as structural probes.

Adenosine Triphosphate↗