Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform.
Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.
Wang
Lijun
L
Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA.
Cowley
Aaron B
AB
Benson
David R
DR
D020816:Amino Acid Motifs; D001051:Apoenzymes / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D020134:Catalytic Domain; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology*; D051336:Mitochondrial Membranes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation / Q000187:drug effects; D012694:Serine / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D001051:Apoenzymes; D020035:Holoenzymes; D007527:Isoenzymes; D005973:Glutamine; D012694:Serine; D006639:Histidine; D014508:Urea; D015786:Cytochromes b5; D001120:Arginine