Biomedical subjects

M H Thi

Publications and source records attributed to M H Thi.

5 recordsLinked to original sources

PubMed1999

The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures.

The molecular mechanisms that evolution has been employing to adapt to environmental temperatures are poorly understood. To gain some further insight into this subject we solved the crystal structure of triosephosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima (TmTIM). The enzyme is a tetramer, assembled as a dimer of dimers, suggesting that the tetrameric wild-type phosphoglycerate kinase PGK-TIM fusion protein consists of a core of two TIM dimers covalently linked to 4 PGK units. The crystal structure of TmTIM represents the most thermostable TIM presently known in its 3D-structure. It adds to a series of nine known TIM structures from a wide variety of organisms, spanning the range from psychrophiles to hyperthermophiles. Several properties believed to be involved in the adaptation to different temperatures were calculated and compared for all ten structures. No sequence preferences, correlated with thermal stability, were apparent from the amino acid composition or from the analysis of the loops and secondary structure elements of the ten TIMs. A common feature for both psychrophilic and T. maritima TIM is the large number of salt bridges compared with the number found in mesophilic TIMs. In the two thermophilic TIMs, the highest amount of accessible hydrophobic surface is buried during the folding and assembly process.

Amino Acid Sequence↗

PubMed1996

ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions.

CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (Km >/=200 microM), and the peptide bond turnover number was approximately 10 min-1. CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the alpha-helical secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity. CcdA lost alpha-helical structure at elevated temperatures (Tm approximately 50 degrees C), and its degradation became independent of ATP hydrolysis at this temperature. ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.

ATP-Dependent Proteases↗

PubMed1996

Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme.

The Camelidae is the only taxonomic family known to possess functional heavy-chain antibodies, lacking light chains. We report here the 2.5 A resolution crystal structure of a camel VH in complex with its antigen, lysozyme. Compared to human and mouse VH domains, there are no major backbone rearrangements in the VH framework. However, the architecture of the region of VH that interacts with a VL in a conventional FV is different from any previously seen. Moreover, the CDR1 region, although in sequence homologous to human CDR1, deviates fundamentally from the canonical structure. Additionally, one half of the CDR3 contacts the VH region which in conventional immunoglobulins interacts with a VL whereas the other half protrudes from the antigen binding site and penetrates deeply into the active site of lysozyme.

Amino Acid Sequence↗

PubMed1993

Purification, circular dichroism analysis, crystallization and preliminary X-ray diffraction analysis of the F plasmid CcdB killer protein.

Large crystals of the Escherichia coli F plasmid CcdB killer protein were grown from solutions containing 32% ammonium sulphate. The crystals belong to space group P4(2)2(1)2 with a = b = 104.52 A and c = 88.45 A or P2(1)2(1)2(1) with a = 77.62 A, b = 93.28 A and c = 141.44 A. Both crystal forms diffract to 2.6 A resolution. Structure determination by multiple isomorphous replacement is under way.

Bacterial Proteins↗

PubMed1993

Purification, crystallization, and preliminary X-ray studies on the rhizome lectin from stinging nettle and its complex with NN'N"-triacetylchitotriose.

Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN'N"-triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P2(1)2(1)2(1) with unit cell dimensions a = 54.3 (1) A, b = 62.2 (1) A, and c = 92.4 (2) A, and diffracts to 3.0 A resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P2(1)2(1)2(1) with cell constants a = 37.69 (4) A, b = 48.97 (6) A, and c = 57.32 (4) A. These crystals diffract to at least 2.0 A resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way.

Binding Sites↗