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Insertion of the cytochrome b₅ heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome’s architecture

Department of Chemistry and the Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, Pennsylvania 16802, USA

(RECEIVED May 28, 2004; FINAL REVISION August 9, 2004; ACCEPTED August 10, 2004)

Under native conditions, apocytochrome b₅ 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 T_m of 8.3°C, a C_m of 1.5 M urea, and a G° of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the ~6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b₅ suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.

Keywords: protein structure/folding; stability and mutagenesis

Abbreviations: , change in chemical shift in ppm • bp, base pair • CD, circular dichroism • DQF-COSY, double-quantum-filtered correlated spectroscopy • EbE1, chimeric protein whose N- and C-terminal stretches are composed of residues from PsaE and whose intervening residues come from cytochrome b₅ • EDTA, ethylenediaminetetraacetic acid • IPTG, isopropylthio--D-galactoside • LB, Luria-Bertani medium • MRE, molar residual ellipticity • NOE, nuclear Overhauser effect • NOESY, two-dimensional nuclear Overhauser effect spectroscopy • OD₆₀₀, optical density at 600 nm • PAGE, polyacrylamide gel electrophoresis • PDB, Protein Data Bank • PMSF, phenylmethylsulfonyl fluoride • PsaE, photosystem I accessory protein E • S7002, Synechococcus sp. PCC 7002 • SDS, sodium do-decylsulfate • SH3, Src homology domain 3 • TOCSY, totally correlated two-dimensional spectroscopy • Tris, tris(hydroxymethyl)aminomethane

Reprint requests to: Juliette T.J. Lecomte, Chemistry Department, The Pennsylvania State University, 152 Davey Laboratory, University Park, PA 16802, USA; e-mail: jtl1{at}psu.edu; fax: (814) 863-8403.

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