A4 I2
Biochemical Society Transactions (2000) Volume 28, Part 5
493 Complementation of AclpB mutation defect by pClpB and sup-
495 Exploration of conformational space of small biological com-
pression of the defect by overproduction of Hsps: GroELIGroES or DnaIWDnaJIGrpE E.Matuszewska. S. Kedzierska, A. Lagodzinska and A. Taylor University of Gdansk, Department of Biochemistry, Kladki 24,80-822 Gdansk, Poland
pounds. Michael E. Popo~,Ilya V. Kashparov, Sergey N. Ruzheinikov' IBCh, Miklukho-Makkaya 16/10, Moscow, 117871, Russia; *University of Sheffield, Firth Court, Western Bank, Sheffield SlO 2TN, UK;e-mail:
[email protected]
Submission of E.coli cells to heat shock (30"C, 4 5 T , 15 min) causes an intracellular aggregation of endogenous proteins. These aggregates form a distinct fraction, denoted S, which is separable by sucrose-density-gradient centrifugation. The S fraction in wild type (wt) cells disappeared in 10 min after the culture was transferred to 37°C for recovery [I]. The S fraction removal was significantly retarded in the AclpB mutant cells: 60% of the S fraction still remained in the MC4100 clpB::kan mutant and 65% in MClOOO clpB::kan at the 4!ith min of the experiment. Thus the efect of the mutation was comparable in the two strains. Experiments on complementation and suppression of the AclpB mutation were based on this phenotypic feature. Introduction of pClpB into MC4100clpB::kan resulted in complete removal of the S fraction at the 3!ith min of the experiment (20 min recovery at 37"C), whereas 34% of the S fraction was still present at the 45th min in MC1000clp::kan cells. The two strains differ genotypically. Looking for an explanation of the difference in the results of the complementation test a comparison of ClpB levels in these strains was performed based on gel electrophoresis and densitometry. Overproduction of ClpB from the same plasmid reached different levels in the two strains. It was approximately 2.5-fold and &fold in MC4100 and MClOOO respectively (though it was not clear why). The complementation appeared to be dependent on the level of ClpB. However, high levels of overproduction of ClpB reduced efficiency of S fraction removal. The overproduction of either GroEL/GroES or DnaK/DnaJ/GrpE not only compensated for the defect caused by a AclpB mutation but entirely prevented S fraction accumulation, following heat shock. [lIKucharczyk et al., (1991) Mol. Microbio1.52935-2945.
1494 Study of conformation changes in hemoglobin A using cationic and anionic surfactants a n d Mostafa, Rezaei-Tavirani IIam University of Medical Sciences, Ilam, Iran Hemoglobins are the most widespread respiratory pigments in animal kingdom. The conformational changes of hemoglobin A is studied by ligand-binding method using sevral anionic and cationic surfactants in 2.5 mM phosphate buffer and p H 6.4. The thermodynamic parameters such as molar free energy changes, molar enthalpy changes and molar entropy changes are calculated. I n this study it is shown that denaturation of hemoglobin in the presence of different concentrations of denaturants is a muliphasic process. The role of ionic and hydrophobic forces for the each phases of denaturation of hemoglobin is also studied.
0 2000 Biochemical Society
Solving biological problems like protein docking, modelling of mutations etc is in increasing need of improvements in protein modelling. An important task in prediction of a protein StrUCNre is generation of a full rotameric library of amino acids. Building such a library by analyzing known 3D structures has some disadvantages. For example, unusual (e.g. ornithin) or chemically modified (e.g. glycosylated or phosphorylated) amino acids are not well presented in structure data bases. Here we present a fragment based approach which allows exploration of a full conformational space of an amino acid. The key principle of this approach is incremental stepwise elongation of the peptide chain including systematic variation of the overlapping pairs of corresponding dihedral angles. The effect of 'conformational explosion' is avoided by rejecting intermediate conformations with high relative energy on each calculation step. Furthermore, the approach presented allows very accurate scanning of the conformational space aimed to find a11 energy minima allowed for a given compound of interest. This algorithm is implemented as a module in the novel computational software for molecular modelling called eFold. The method presented proved to be useful for building the rotamer libraries of the free amino acids as well as for reconstructing the amino acid side chains in proteins. The completeness of our rotamer library for common amino acids has been proved by comparing it with a library obtained by the analysis of the vast number of high resolution protein structures presented in the Protein Data Bank.
1496 Transthyretin
Stability
in
Familial
Amyloidotic
Polyneuropathy A. Ouintas, D. Vaz, M.J. Saraiva, R.M.M. Brito, Azinhaga de Sta. Cornba, Ed. IBILI
