[Thermal denaturation of eukaryotic class 1 translation termination factor eRF1. Relationship between stability of the eRF1 molecule and alteration of functional activity of its mutants].
Thermal denaturation of eukaryotic class-1 translation termination factor eRF1 and its mutants was examined using differential scanning microcalorimetry (DSK). Changes of free energy caused by mutants in the N domain of human eRF1 were calculated. Melting of eRF1 molecule composed of three individual domains is cooperative. Some amino acid substitutions did not affect protein thermostability and in some other cases even slightly stabilize the protein globule. These imply that these amino acid residues are not involved in maintenance of the 3D structure of human eRF1. Thus, in Glu55Asp, Tyr125Phe, Asn61Ser, Glu55Arg, Glu55A1a, Asn61Ser + Ser64Asp, Cys127Ala and Ser64Asp mutants selective inactivation of release activity is not caused by a destabilization of protein 3D structure and, most likely, is associated with local stereochemical changes introduced by substitutions of amino acid side chains in the functionally essential sites of N-domain molecule. Some residues (Asn129, Phe131) as shown by calorimetric measurements are essential for preservation of stable protein structure, but at the same time they affect selective stop codon recognition probably via their neighboring amino acids. Recognition of UAG and UAA stop codons in vitro is more sensitive to preservation of protein stability than the UGA recognition.
Mit'kevich
V A
VA
Kononenko
A V
AV
Oparina
N Iu
NIu
Kolosov
P M
PM
Makarov
A A
AA
Kiselev
L L
LL
D019943:Amino Acid Substitution; D002152:Calorimetry, Differential Scanning; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D044366:Transition Temperature
C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D011994:Recombinant Proteins