Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding.


The pairing of two alpha-helices at opposite ends of the chain is a highly conserved structural motif found throughout the cytochrome c family of proteins. It has previously been shown that association of the N- and C-terminal helices is a critical early event in the folding process of horse cytochrome c and is responsible for the formation of a partially folded intermediate (INC). In order to gain further insight into the structural and energetic basis of helix packing interactions and their role in folding, we prepared a series of horse cytochrome c variants in which Leu94, a critical residue at the helix contact site, was replaced by Ile, Val, or Ala. The Ile and Val substitutions resulted in minor changes in the stability of the native state, indicating that conservative mutations can be accommodated at the helix interface with only minor structural perturbations. In contrast, the L94A mutation resulted in a 3.5 kcal/mol decrease in unfolding free energy, suggesting that the smaller Ala side chain causes severe packing defects at the helix interface. The effect of these mutations on the kinetics of folding and unfolding as a function of denaturant concentration was studied by a systematic series of stopped-flow fluorescence measurements. The proteins with Leu, Ile, or Val at position 94 exhibit a major unresolved fluorescence change during the 1-ms dead time of the stopped-flow refolding measurements, while this effect is less pronounced in L94A, indicating that the rapid formation of a compact state (IC) with largely quenched Trp59 fluorescence is favored by a large hydrophobic side chain at the helix-helix interface. Despite their small effects on overall stability, the L94I and L94V mutations result in a substantial reduction in the relative amplitude of the fastest observable folding phase (formation of INC) consistent with a strong decrease in the population of INC compared to the wild-type protein. This effect is amplified in the case of the destabilizing L94A variant, which exhibits slower folding kinetics and negligible accumulation of INC. Whereas the presence of a large hydrophobic side chain at position 94 is sufficient for the stabilization of IC, the subsequent partially folded intermediate, INC, is stabilized by specific interactions that are responsible for the proper packing of the two alpha-helices. Study holds ProTherm entries: 5024, 5025, 5026, 5027 Extra Details:

Submission Details

ID: xRjWzi7N4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Colón W;Elöve GA;Wakem LP;Sherman F;Roder H,Biochemistry (1996) Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding. PMID:8611545
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2B4Z 2005-09-27T00:00:00+0000 1.5 Crystal structure of cytochrome C from bovine heart at 1.5 A resolution.
2YBB 2011-03-02T00:00:00+0000 19.0 Fitted model for bovine mitochondrial supercomplex I1III2IV1 by single particle cryo-EM (EMD-1876)
3J2T 2012-12-23T00:00:00+0000 9.5 An improved model of the human apoptosome
5C0Z 2015-06-12T00:00:00+0000 1.12 The structure of oxidized rat cytochrome c at 1.13 angstroms resolution
5C9M 2015-06-28T00:00:00+0000 1.36 The structure of oxidized rat cytochrome c (T28A) at 1.362 angstroms resolution.
5DF5 2015-08-26T00:00:00+0000 1.3 The structure of oxidized rat cytochrome c (T28E) at 1.30 angstroms resolution.
5JUY 2016-05-10T00:00:00+0000 4.1 Active human apoptosome with procaspase-9
6FF5 2018-01-03T00:00:00+0000 1.74 X-ray structure of bovine heart cytochrome c at high ionic strength
6N1O 2018-11-09T00:00:00+0000 1.55 Oxidized rat cytochrome c mutant (S47E)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.3 Cytochrome c P00021 CYC_COLLI
92.2 Cytochrome c P00020 CYC_ANAPL
92.3 Cytochrome c P81280 CYC_ALLMI
90.5 Cytochrome c Q52V10 CYC_SAISC
93.3 Cytochrome c P00012 CYC_MIRLE
93.3 Cytochrome c Q52V09 CYC_CEPBA
93.3 Cytochrome c P00013 CYC_MINSC
93.3 Cytochrome c P00014 CYC_MACGI
94.3 Cytochrome c P00011 CYC_CANLF
94.3 Cytochrome c P62898 CYC_RAT
94.3 Cytochrome c P00008 CYC_RABIT
94.3 Cytochrome c P62897 CYC_MOUSE
95.2 Cytochrome c P68098 CYC_LAMGU
95.2 Cytochrome c P68100 CYC_ESCRO
95.2 Cytochrome c P68099 CYC_CAMDR
94.3 Cytochrome c P00007 CYC_HIPAM
97.1 Cytochrome c P62896 CYC_SHEEP
97.1 Cytochrome c P62895 CYC_PIG
97.1 Cytochrome c P62894 CYC_BOVIN
99.0 Cytochrome c P68096 CYC_EQUBU
99.0 Cytochrome c P68097 CYC_EQUAS
100.0 Cytochrome c P00004 CYC_HORSE
90.3 Cytochrome c B4USV4 CYC_OTOGA