Propensities of aromatic amino acids versus leucine and proline to induce residual structure in the denatured-state ensemble of iso-1-cytochrome c.
Histidine-heme loop formation in the denatured state of a protein is a sensitive means for probing residual structure under unfolding conditions. In this study, we use a host-guest approach to investigate the relative tendencies of different amino acids to promote residual structure under denaturing conditions. The host for this work is a 6-amino-acid insert of five alanines, followed by a lysine engineered immediately following a unique histidine near the N-terminus of yeast iso-1-cytochrome c. We substitute the fourth alanine in this sequence HAAAXAK (with X=Trp, Phe, Tyr, and Leu). The effects of proline are tested with substitutions at positions 1 and 5 in the insert (HPAAAAK and HAAAAPK, respectively). Thermodynamic studies on His-heme loop formation in 3 M guanidine hydrochloride reveal significant stabilization of residual structure by aromatic amino acids, particularly Trp and Phe, and minimal stabilization of residual structure by Leu. Prolines slightly disfavor His-heme loop formation, presumably due to enhanced chain stiffness. Kinetic studies reveal that much of the change in His-heme loop stability for the aromatic amino acids is caused by a slowdown in the rate of His-heme loop breakage, indicating that residual structure is preferentially stabilized in the closed-loop form of the denatured state.
Finnegan
Michaela L
ML
Department of Chemistry and Biochemistry, Biochemistry Program and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA.
Bowler
Bruce E
BE
D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
D024322:Amino Acids, Aromatic; C014059:CYC1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D011392:Proline; D007930:Leucine