Rat liver betaine-homocysteine S-methyltransferase equilibrium unfolding: insights into intermediate structure through tryptophan substitutions.
Equilibrium folding of rat liver BHMT (betaine-homocysteine methyltransferase), a TIM (triosephosphate isomerase)-barrel tetrameric protein, has been studied using urea as denaturant. A combination of activity measurements, tryptophan fluorescence, CD and sedimentation-velocity studies suggested a multiphasic process including two intermediates, a tetramer (I4) and a monomer (J). Analysis of denaturation curves for single- and six-tryptophan mutants indicated that the main changes leading to the tetrameric intermediate are related to alterations in the helix alpha4 of the barrel, as well as in the dimerization arm. Further dissociation to intermediate J included changes in the loop connecting the C-terminal alpha-helix of contact between dimers, disruption of helix alpha4, and initial alterations in helix alpha7 of the barrel, as well as in the dimerization arm. Evolution of the monomeric intermediate continued through additional perturbations in helix alpha7 of the barrel and the C-terminal loop. Our data highlight the essential role of the C-terminal helix in dimer-dimer binding through its contribution to the increased stability shown by BHMT as compared with other TIM barrel proteins. The results are discussed in the light of the high sequence conservation shown by betaine-homocysteine methyltransferases and the knowledge available for other TIM-barrel proteins.
Garrido
Francisco
F
Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Arturo Duperier 4, 28029 Madrid, Spain.
Gasset
María
M
Sanz-Aparicio
Juliana
J
Alfonso
Carlos
C
Pajares
María A
MA
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D050940:Betaine-Homocysteine S-Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D014364:Tryptophan; D050940:Betaine-Homocysteine S-Methyltransferase