title: Characterization of an independent structural unit in apocytochrome b5.
abstract: Apocytochrome b5 is a partially folded protein which contains a stable structural unit under native conditions [Moore, C.D., Al-Misky, O.N., & Lecomte, J.T.J. (1990) Biochemistry 30, 8357-8365]. In this work, the fold of the unit was examined by using 1H and 15N-edited two-dimensional NMR spectroscopy. It was found that it contains four of the five beta-strands and two of the six alpha-helices present in the holoprotein. The remainder of the structure appears to be mostly unstructured and fluctuating among several conformations. The structural unit is stabilized by a hydrophobic core formed by residues from each of the folded elements of secondary structure. Nuclear Overhauser effects and chemical shift values demonstrated that the unit is structurally similar in the apo- and holoproteins. However, the backbone amide hydrogen exchange was found to be much accelerated in the apoprotein. The paramagnetic relaxation agent HyTEMPO was used to probe the packing of the structure. HyTEMPO has unrestricted access to the empty heme binding site whereas it is unable to penetrate the stabilizing core. It was concluded that addition of the heme is necessary for the last strand to dock properly to the rest of the sheet. The kinetics of refolding of the apoprotein were monitored by stopped-flow fluorescence spectroscopy. Extensive protection of the sole tryptophan residue by docking of the two polypeptide termini occurs in less than 60 ms. It was proposed that apocytochrome b5, with its two-region behavior, might serve as a model for the design of proteins which bind a prosthetic group.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802.
authors.forename: C D
authors.initials: CD
authors.lastname: Moore
authors.forename: J T
authors.initials: JT
authors.lastname: Lecomte
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D001665:Binding Sites; D003497:Cyclic N-Oxides; D003573:Cytochrome b Group / Q000737:chemistry*; D045303:Cytochromes b; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D014508:Urea
chemical_list: D001059:Apoproteins; D003497:Cyclic N-Oxides; D003573:Cytochrome b Group; D013113:Spin Labels; D006418:Heme; D014508:Urea; D045303:Cytochromes b; C001803:tempol