Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture.
Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.
Chemistry Department, The Pennsylvania State University, 152 Davey Laboratory, University Park, PA 16802, USA.
Jane A
JA
Knappenberger
Christina M
CM
Kraemer-Pecore
Juliette T J
JT
Lecomte
D001665:Binding Sites; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D006418:Heme; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013057:Spectrum Analysis; D046940:Synechococcus / Q000737:chemistry; D013816:Thermodynamics; D018909:src Homology Domains / Q000235:genetics
D045331:Photosystem I Protein Complex; D011993:Recombinant Fusion Proteins; C079741:photosystem I, psaE subunit; D006418:Heme; D015786:Cytochromes b5