A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.
The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.
Department of Chemistry and the Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park 16802, USA.
A J
AJ
Constans
M R
MR
Mayer
S F
SF
Sukits
J T
JT
Lecomte
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics; D045303:Cytochromes b; D005453:Fluorescence; D006418:Heme / Q000737:chemistry*; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D014364:Tryptophan / Q000737:chemistry
D001059:Apoproteins; D003573:Cytochrome b Group; D006418:Heme; D014364:Tryptophan; D045303:Cytochromes b