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

Abstract

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.

Local Protein Structures

“…They may be directly associated with protein function [191][192][193][194][195] and folding [192]. An important number of studies focused on the cytochrome c and the use of compatible Ω-loops to replace existing local 3D conformations [191,[196][197][198]. These studies have to be viewed as complementary to investigations on closed loops, Tight End Fragment (TEF) and MIR (Most Interacting Residues) which define loop fragments that are able in three-dimensional (3D) space to nearly close their ends [199][200][201].…”

Loop Entropy and Cytochrome c Stability

“…Mutant proteins in which loop D is swapped with loops from other cytochromes c contain apo protein but no holo protein. 17 The sensitivity to mutation suggests that loop D may be involved in apo protein recognition by the heme lyase that catalyzes heme attachment. Mutational substitutions in loop D have identified amino acid preferences possibly related to lyase recognition.…”

“…Mutational substitutions in loop D have identified amino acid preferences possibly related to lyase recognition. 17,18 On the basis of the genetic studies, loop A was chosen as a target for polypeptide insertions. Inspection of three-dimensional molecular models of iso-2 led to selection of the Gly23-Gly24 site within loop A.…”

Rose is a rose is a rose. Especially if you're a George

“…This initial work also laid the groundwork for much subsequent analysis of the role of omega (and other loops) in protein structure, function, and dynamics (see, for example, see Refs. 46–64). George has recently applied methods similar to those he used to classify and categorize proteins (what we call a “George‐like analysis”) to RNA structure and RNA structure formation 65–67.…”