High Resolution Low Temperature Spectrophotometry of Cytochromes c

Elliott, W.B.; Margoliash, E.

doi:10.1007/978-1-4757-0782-3_5

Cited by 4 publications

(3 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Low temperature spectroscopy of intact yeast cells provides a quantitative measure of how much holo-cytochrome c is produced within the cell (Elliott & Margoliash, 1971;Cottrell et al, 1975;Schweingruber et al, 1977). Holo-cytochrome c is defined as a cytochrome c polypeptide that has entered the mitochondria, has been covalently linked to a heme group, and has folded properly.…”

Section: Amount Of Folded Cytochrome C Present In Mutant Yeast Strainsmentioning

confidence: 99%

“…1989;Auld & Pielak, 1991;Fumo et a!., 1995) and the facility with which the protein can be purified (Sherman et a!., 1968). In this facultative aerobe, growth curves in media containing nonfermentable carbon sources can identify functional properties of variant iso-l-cytochromes c (Ernst et al, 1985;Fetrow et a!., 1989) and low temperature spectroscopy of intact yeast cells grown on fermentable carbon sources (Elliott & Margoliash, 1971;Cottrell et a!., 1975;Schweinpber et al, 1977) can detect structural properties of the protein in vivo; consequently, folded, but nonfunctional, proteins can be identified without resorting to protein purification. Further, a number of crystal and solution structures of eukaryotic cytochromes c have been solved (Tanaka et al, 1975;Takano & Dickerson, 1981;Ochi et al, 1983;Bushnell et al, 1990;Murphy et al, 1992;Qi et al, 1994;Baistrocchi et al, 1996;Qi et al, 1996;Banci et al, 1997) and many mutant proteins have been crystallized and their structures solved (Hampsey et al, 1986;Louie et al, 1988;Langen et a!., 1992;Murphy et al, 1993;Berghuis et al, 1994;Lo et al, 1995).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mutagenesis of histidine 26 demonstrates the importance of loop‐loop and loop‐protein interactions for the function of iso‐1‐cytochrome c

et al. 1998

View full text Add to dashboard Cite

In yeast iso-I-cytochrome c, the side chain of histidine 26 (His26) attaches omega loop A to the main body of the protein by forming a hydrogen bond to the backbone atom carbonyl of glutamic acid 44. The His26 side chain also forms a stabilizing intra-loop interaction through a hydrogen bond to the backbone amide of asparagine 3 1 . To investigate the importance of loop-protein attachment and intra-loop interactions to the structure and function of this protein, a series of site-directed and random-directed mutations were produced at His26. Yeast strains expressing these variant proteins were analyzed for their ability to grow on non-fermentable carbon sources and for their intracellular production of cytochrome c. While the data show that mutations at His26 lead to slightly decreased intracellular amounts of cytochrome c, the level of cytochrome c function is decreased more. The data suggest that cytochrome c reductase binding is affected more than cytochrome c oxidase or lactate dehydrogenase binding. We propose that mutations at this residue increase loop mobility, which, in turn, decreases the protein's ability to bind redox partners.

show abstract

Section: Amount Of Folded Cytochrome C Present In Mutant Yeast Strainsmentioning

confidence: 99%

mentioning

confidence: 99%

Mutagenesis of histidine 26 demonstrates the importance of loop‐loop and loop‐protein interactions for the function of iso‐1‐cytochrome c

et al. 1998

View full text Add to dashboard Cite

show abstract

“…Because S. cerevisiae requires a functional cytochrome c to grow on nonfermentable carbon sources, function can be assayed by growth of mutant yeast strains on lactate, glycerol, or ethanol media (12). The presence of holoprotein, cytochrome c to which the heme group is covalently attached, can be detected by whole cell difference spectroscopy following growth of cells in dextrose medium, regardless of the functionality of the pro-tein (12,13). A lack of holoprotein would indicate that a mutation has altered the fold or stability of apo-or holoprotein so that it is more readily proteolyzed or that it has disrupted a step in the biosynthetic processing of holocytochrome c, such as transcription in the nucleus, translation on free ribosomes, mitochondrial recognition and import, or recognition by cytochrome c heme lyase, the protein that covalently attaches heme to the apoprotein (14,15).…”

mentioning

confidence: 99%

Loop Replacement and Random Mutagenesis of -Loop D, Residues 70 84, in Iso-1-cytochrome c

Mulligan-Pullyblank¹,

Spitzer

Gilden

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

To study the role of omega loop D, residues 70 -84, in the structure and function of yeast iso-1-cytochrome c, this loop was replaced with homologous and heterologous loops. A novel method was developed for rapid insertion of these mutations into the yeast chromosome at the CYC1 locus. The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins are nonfunctional. Whole cell difference spectroscopy shows that no holocytochrome c is present; however, apoprotein is found by immunoblot analysis. Thus, apoprotein is present in these mutant strains, but it cannot bind heme and cannot compete with wild type apoprotein conversion to holoprotein. This is a unique example of a set of loop replacements that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significant role in cytochrome c biosynthesis in vivo. To identify the significant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79, was accomplished. Sequencing of the random mutants shows that strict conservation of none of these residues is required to produce a minimally functional cytochrome c. Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and ␤-branched amino acids at position 78. Implications for the role of loop D in the structure and function of iso-1-cytochrome c are discussed.The protein folding problem is an important, unsolved problem in molecular biology. An understanding of this problem is important to utilization of the data being generated by the human genome project and to further the design and engineering of proteins with specific structures and functions. Loops and turns, the connections between the regular secondary structures, are critical to the design of properly folded proteins. These structures are difficult to characterize because of their range of conformations. The short ␤-turns and reverse turns were the first to be characterized (1, 2); then the longer ⍀-loops were described (3); and finally a more comprehensive definition of loops was published (Ref. 4; for review, see Ref. 5). We are interested in defining the role that loops, particularly ⍀-loops, play in protein folding, function, and stability.⍀-Loops are nonregular protein secondary structures defined as a sequence of six or more residues with a short distance between segment termini and a lack of regular hydrogen bonding between backbone residues (3). However, significant hydrogen bonding and hydrophobic interactions can exist between side chain and backbone atoms within the loop itself.

show abstract

Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

Cartling

Wilbrandt

1981

Biochimica et Biophysica Acta (BBA) - Bioenergetics

View full text Add to dashboard Cite

High Resolution Low Temperature Spectrophotometry of Cytochromes c

Cited by 4 publications

References 9 publications

Mutagenesis of histidine 26 demonstrates the importance of loop‐loop and loop‐protein interactions for the function of iso‐1‐cytochrome c

Mutagenesis of histidine 26 demonstrates the importance of loop‐loop and loop‐protein interactions for the function of iso‐1‐cytochrome c

Loop Replacement and Random Mutagenesis of -Loop D, Residues 70 84, in Iso-1-cytochrome c

Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

Contact Info

Product

Resources

About