Influence of point mutations on the flexibility of cytochrome b₅: Molecular dynamics simulations of holoproteins

Abstract

Two membrane-bound isoforms of cytochrome b5 have been identified in mammals, one associated with the outer mitochondrial membrane (OM b5) and the other with the endoplasmic reticulum (microsomal, or Mc b5). The soluble heme binding domains of OM and Mc b5 have highly similar three-dimensional structures but differ significantly in physical properties, with OM b5 exhibiting higher stability due to stronger heme association. In this study, we present results of 8.5-ns length molecular dynamics simulations for rat Mc b5, bovine Mc b5, and rat OM b5, as well as for two rat OM b5 mutants that were anticipated to exhibit properties intermediate between those of rat OM b5 and the two Mc proteins: the A18S/I32L/L47R triple mutant (OM3M) and the A18S/I25L/I32L/L47R/L71S quintuple mutant (OM5M). Analysis of the structure, fluctuations, and interactions showed that the five b5 variants used in this study differed in organization of their molecular surfaces and heme binding cores in a way that could be used to explain certain experimentally observed physical differences. Overall, our simulations provided qualitative microscopic explanations of many of the differences in physical properties between OM and Mc b5 and two mutants in terms of localized changes in structure and flexibility. They also reveal that opening of a surface cleft between hydrophobic cores 1 and 2 in bovine Mc b5, observed in two previously reported simulations (E. M. Storch and V. Daggett, Biochemistry, 1995, Vol. 34, pp. 9682-9693; A. Altuve, Biochemistry, 2001, Vol. 40, pp. 9469-9483), probably resulted from removal of crystal contacts and likely does not occur on the nanosecond time scale. Finally, the MD simulations of OM5M b5 verify that stability and dynamic properties of cytochrome b5 are remarkably resistant to mutations that dramatically alter the stability and structure of the apoprotein.

Direct detection of malaria infected red blood cells by surface enhanced Raman spectroscopy

“…The Δν = 646 and 723 cm −1 peaks are characteristic peaks for lipid according to Drescher 40 , while the Δν = 1460 and 1712 cm −1 peaks result from vibrational modes in proteins. In addition, i 32 also increased significantly at peaks Δν = 951, 1089, 1124, 1247, 1330 and 1377 cm −1 , which represent the structure change of membrane lipids and proteins 28,3033,41 . However, $i_{1599}^{32}$ (≈ 2.6) stayed almost the same as $i_{1599}^{24}$, which indicated that the peak at Δν = 1599 cm −1 is the most appropriate SERS peak to characterize the ring stage iRBCs.…”

“…1 are from these major membrane components, and the detailed peak assignments are listed in Table 1. The characteristic peaks at Δν = 421, 519, 957, 1064, 1124, 1330 and 1379 cm −1 are assigned to the vibrational mode of lipids 28–31 , peaks at Δν = 749, 1258, 1552 and 1618 cm −1 represent vibrational modes of membrane proteins 30,32,33 , and peaks at Δν = 840 and 898 cm −1 could be assigned to saccharide bands 34 .…”

Molecular modeling of cytochrome b 5 with a single cytochrome c-like thioether linkage

“…showed that an open-closed cleft transition occurs in this region for bovine and other cyt b 5 species [24,26,28].…”

“…To date, plenty of theoretical studies have been performed for cyt b 5 [24][25][26][27][28][29][30][31][32], cyt b 562 [33][34][35][36], and cyt c [37][38][39]; these studies have aimed to gain structural insights, as well as to elucidate the mechanisms of holoprotein maturation. Using molecular modeling in combination with MD simulations, we recently studied the structural consequences of introducing a histidine tag, the (His) 6 tag, to the N-terminus of bovine Mc cyt b 5 [32].…”

Studies of Interaction Between Tetrabutylammonium Bromide Based Deep Eutectic Solvent and Dna Using Fluorescence Quenching Method and Circular Dichroism Spectroscopy

“…The potentials of DNA were increasingly recognized especially in the applications of biosensors, catalytic membranes, absorbing membranes and support for catalysts [1,2]. However, DNA is unstable in aqueous solution, resulting in denaturation of nucleic acid structure after 1 month of storage at ambient temperature [3].…”