Structural and thermodynamic encoding in the sequence of rat microsomal cytochrome b(5).
The water-soluble domain of rat microsomal cytochrome b(5) is a convenient protein with which to inspect the connection between amino acid sequence and thermodynamic properties. In the absence of its single heme cofactor, cytochrome b(5) contains a partially folded stretch of 30 residues. This region is recognized as prone to disorder by programs that analyze primary structures for such intrinsic features. The cytochrome was subjected to amino acid replacements in the folded core (I12A), in the portion that refolds only when in contact with the heme group (N57P), and in both (F35H/H39A/L46Y). Despite the difficulties associated with measuring thermodynamic quantities for the heme-bound species, it was possible to rationalize the energetic consequences of both types of replacements and test a simple equation relating apoprotein and holoprotein stability. In addition, a phenomenological relationship between the change in T(m) (the temperature at the midpoint of the thermal transition) and the change in thermodynamic stability determined by chemical denaturation was observed that could be used to extend the interpretation of incomplete holoprotein stability data. Structural information was obtained by nuclear magnetic resonance spectroscopy toward an atomic-level analysis of the effects.
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA. lecomte_jtj@jhu.edu
Juliette T J
JT
Lecomte
Kunal
K
Mukhopadhyay
Matthew P
MP
Pond
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics; D015786:Cytochromes b5 / Q000235:genetics* / Q000502:physiology; D004795:Enzyme Stability; D014644:Genetic Variation; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000201:enzymology*; D008958:Models, Molecular; D015394:Molecular Structure; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D006418:Heme; D015786:Cytochromes b5