Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c.
As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., & Nall, B. T. (1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)
Wood
L C
LC
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
White
T B
TB
Ramdas
L
L
Nall
B T
BT
D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D005998:Glycine; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D011392:Proline; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D003574:Cytochrome c Group; D006146:Guanidines; C032139:iso-2-cytochrome C; D045304:Cytochromes c; D011392:Proline; D019791:Guanidine; D005998:Glycine