Linked Life Data

12664263

Source:http://linkedlifedata.com/resource/pubmed/id/12664263

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2003-3-28
pubmed:abstractText
The histone-like DNA-binding proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability. We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU protein from the hyper-thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C). The structural data presented clearly show that the HU Tmar has a fold similar to its thermophilic homologue HU from Bacillus stearothermophilus (HU Bst). Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar. Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme thermophilic protein HU Tmar to mesophilic, like HU Bsu. In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu. The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type. The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution. A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa. Our analysis points to this as the prime cause of the destabilization compared to the wild type. The three new structures were compared, together with the X-ray structure of a similar protein, HU Bst, with the aim of relating their structural properties and different thermal stability. The presented results show that the HU Tmar protein achieves its stability by employing a dual strategy. On the one hand, we observe local hydrophobic interactions, which stabilize the secondary structure elements, and on the other hand, electrostatic interactions between side chains.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1431-0651
pubmed:author
pubmed:issnType
Print
pubmed:volume
7
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
111-22
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed-meshheading:12664263-Amino Acid Sequence, pubmed-meshheading:12664263-Bacillus subtilis, pubmed-meshheading:12664263-Bacterial Proteins, pubmed-meshheading:12664263-Base Sequence, pubmed-meshheading:12664263-Crystallography, X-Ray, pubmed-meshheading:12664263-DNA, Bacterial, pubmed-meshheading:12664263-DNA-Binding Proteins, pubmed-meshheading:12664263-Dimerization, pubmed-meshheading:12664263-Drug Stability, pubmed-meshheading:12664263-Geobacillus stearothermophilus, pubmed-meshheading:12664263-Models, Molecular, pubmed-meshheading:12664263-Molecular Sequence Data, pubmed-meshheading:12664263-Mutagenesis, Site-Directed, pubmed-meshheading:12664263-Protein Folding, pubmed-meshheading:12664263-Protein Structure, Secondary, pubmed-meshheading:12664263-Protein Subunits, pubmed-meshheading:12664263-Recombinant Proteins, pubmed-meshheading:12664263-Sequence Homology, Amino Acid, pubmed-meshheading:12664263-Static Electricity, pubmed-meshheading:12664263-Temperature, pubmed-meshheading:12664263-Thermotoga maritima
pubmed:year
2003
pubmed:articleTitle
High-resolution X-ray structure of the DNA-binding protein HU from the hyper-thermophilic Thermotoga maritima and the determinants of its thermostability.
pubmed:affiliation
National and Kapodistrian University of Athens, Faculty of Biology, Department of Biochemistry and Molecular Biology, Panepistimiopolis-Zographou, 157 84 Athens, Greece.
pubmed:publicationType
Journal Article, Comparative Study