Analysis of the structure and stability of omega loop A replacements in yeast iso‐1‐cytochrome c

Abstract

Omega (n)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.

Loop Entropy and Cytochrome c Stability

“…Figure 2 shows a molecular model of iso-2 containing a polyglycine insert. The insert and flanking residues, Gly23-(Gly) 16 -Gly24, are shown in a space-filling representation with the remainder of PWT iso-2 in the framework representation. The loop in Figure 2 is modeled in an extended low energy conformation.…”

“…To improve the accuracy of comparison, the values of DDG(n) are calculated at the T m for the reference protein rather than being extrapolated to a temperature below the transition zone. 8 13,382.7 13,382.9 PWTC(Gly) 12 13,611.3 13,611.1 PWTC(Gly) 16 13,838.0 13,839.3 PWTC(Gly) 20 14,067.2 14,067.6 PWTC(Thr) 4 13,330.5 13,330.5 PWTC(Thr) 8 13,735.5 13,734.5 PWTC(Thr) 12 14,137.7 14,139.5 PWTC(Thr) 16 14,542.4 14,543.5…”

Local Protein Structures

“…For instance, the Ω-loops constitute a particular category characterized by a small distance between their extremities and an important number of contacts in their structure [187][188][189]. They correspond to compact globular loops mainly located at the surface of the proteins [190].…”

Site-specific Reflex Response of Ubiquitin to Loop Insertions

“…6,7 In the other class of studies, loops have been exchanged for a loop from a different protein. 8,9 These loop mutants typically change the length of the loop by no more than a residue or two.…”

“…10 Longer insertions are generally more destabilizing, 5,8 but the decrease in stability per residue is greatest for the first few residues inserted. The structural changes resulting from insertions can often be rationalized based upon new constraints from the altered sequence.…”