Evaluation of Cooperative Interactions between Substructures of Iso-1-Cytochrome c Using Double Mutant Cycles

Abstract

A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through the four cooperative folding units of cytochrome c. The double mutant cycle starts with the iso-1-cytochrome c variant AcTM, a variant with no surface histidines and with asparagine at position 52. Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position. The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle. The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site. Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure. The AcH26I52 variant shows a strong favorable interaction energy, 2.0 +/- 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure. The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.

Kinetics of Loop Formation and Breakage in the Denatured State of Iso-1-cytochrome c

“…50 To prevent intermolecular dimerization during physical studies, all variants contain a Cys102 / Ser mutation. Construction of the H(K2)I52 variant was accomplished by the unique restriction site elimination site-directed mutagenesis as described, 50 using the pBTR1 vector (provided by Grant Mauk at the University of British Columbia) 65 and the unique restriction sites EcoRV and Aat2.…”

“…50 To prevent intermolecular dimerization during physical studies, all variants contain a Cys102 / Ser mutation. Construction of the H(K2)I52 variant was accomplished by the unique restriction site elimination site-directed mutagenesis as described, 50 using the pBTR1 vector (provided by Grant Mauk at the University of British Columbia) 65 and the unique restriction sites EcoRV and Aat2. The iso-1-cytochrome c gene, CYC1, in this vector, was mutated so that Ser at position K5 was reverted to the wild-type residue, Thr, and position 102 was mutated from Thr to Ser to be consistent with other iso-1-cytochrome c variants used in this laboratory.…”

“…We note that previous gdnHCl denaturation experiments have shown that all variants are completely denatured in 3 M gdnHCl. 50 Representative kinetic traces for the AcH89I52 variant, using stopped-flow methods, are shown in Figure 2. The data are for downward pH jumps that lead to loop breakage.…”

Sequence Composition Effects on Denatured State Loop Formation in Iso-1-cytochrome c Variants: Polyalanine versus Polyglycine Inserts

“…8,9,[11][12][13][14][15][16][17][18]46 In previous work, we have noted correlations between denatured state loop stability and global protein stability. 32,33,37 All variants capable of forming a His-heme loop in the denatured state are strongly destabilized relative to the NK5A variant (Table 1) which forms the much less favorable N-terminal amino group-heme loop in the denatured state. How much of this destabilization can be accounted for by differences in denatured state loop stability?…”

“…Lys(-2) terminates each insert sequence. Mutations were made using the unique restriction site elimination method 36 as described37 (for details on protein expression and purification see Supplementary Data).…”