Alternative protein conformations: yeast iso-1-cytochrome c and heme crevice dynamics

The field of protein biochemistry has been dominated by the dogma that a protein sequence yields a 3-dimensional structure important for a singular function. More modern insights are beginning to demonstrate that proteins are not static structures. Rather, proteins undergo numerous conformational fluctuations yielding an ensemble of conformational populations. Conformational change can result in changed or altered protein function. Small or large energetic barriers existing between conformers regulate the ease with which a protein can sample alternative conformations. In the dissertation work presented here, alternative conformations of yeast iso-1-cytochrome c are investigated with particular emphasis on heme crevice loop dynamics. The heme crevice loop, or O-loop D, is a highly conserved, dynamic region. Conformational changes in O-loop D lead to altered electron transfer and peroxidase activity in cytochrome c (Cytc). As Cytc participates in both the electron transport chain and functions as a peroxidase during apoptosis, it is important to understand how this conformational change is regulated. Within O-loop D we investigate the effects of a trimethyllysine to alanine mutation and a destabilizing leucine to alanine mutation at residues 72 and 85, respectively, on heme crevice dynamics. Residue 72 plays an important role in regulating access to alternative heme crevice conformers. Of particular interest, residue 72 plays a role in regulating access to a peroxidase capable conformer of Cytc, a function of Cytc during the early stages of apoptosis. We have also solved the structure of the first monomeric Cytc structure in a peroxidase capable conformer, as well as, a dimeric Cytc structure with CYMAL-6 protruding into the interior of the heme cavity, in a manner potentially similar to the Cytc/cardiolipin interaction

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)