Structural propensities in the heme binding region of apocytochrome b5. I. Free peptides.
The water-soluble domain of rat microsomal cytochrome b5 is a small globular hemoprotein. Under native conditions, the apoprotein consists of a well-folded hydrophobic core and a 42-residue loop, which is substantially disordered in solution. Association with the heme cofactor causes the loop to organize into a second well-folded hydrophobic core encompassing four short helices, H2-H5. Of these, H3 and H4 are recognized as intrinsically disordered by algorithms that analyze primary structures for folding propensities. Three peptides, spanning H2-H5, H2-H3, and H4-H5, were designed, synthesized, and characterized to identify local structural preferences in the isolated loop. In addition, two replacements (D60R and N57P, which are known to stabilize holocytochrome b5) were introduced individually in the H4-H5 peptide. Helical content measured by nuclear magnetic resonance and far-UV circular dichroism spectroscopy in solutions of 2,2,2-trifluoroethanol revealed that H4 possessed a lower propensity to form the holoprotein structure than H3. Both replacements in H4 resulted in measurable changes in observed overall helical propensities. It was concluded that the prediction of intrinsic disorder was reliable. Furthermore, the stability of the holoprotein did not correlate simply with helical propensities in the disordered regions.
Davis
Ronald B
RB
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
Lecomte
Juliette T J
JT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D015786:Cytochromes b5 / Q000737:chemistry*; D006418:Heme / Q000378:metabolism*; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D051381:Rats; D012995:Solubility; D012996:Solutions; D014270:Trifluoroethanol
D010455:Peptides; D012996:Solutions; D006418:Heme; D006639:Histidine; D014270:Trifluoroethanol; D015786:Cytochromes b5