Contribution of the multi-turn segment in the reversible thermal stability of hyperthermophile rubredoxin: NMR thermal chemical exchange analysis of sequence hybrids.
Pyrococcus furiosus (Pf) rubredoxin is the most thermostable protein characterized to date. Reflecting the complications arising from irreversible denaturation of this protein, predictions of which structural regions confer differential thermal stability have utilized kinetic stability measurements, hydrogen exchange protection factors, long range hydrogen bond NMR spin couplings, and molecular dynamics simulations, and have primarily implicated the three-stranded beta-sheet and the adjacent metal binding site. Herein, NMR chemical exchange experiments demonstrate reversible two-state unfolding at the thermal transition temperature (T(m)) for hybrids of Pf and the mesophile Clostridium pasteurianum (Cp) rubredoxins which interchange residues 14-33, the so-called multi-turn segment. This complementary pair of hybrid rubredoxins exhibits largely additive incremental thermal stabilizations vs. the parental proteins. Both stabilization free energy measurements as well as incremental T(m) values indicate that a minimum of 37% of the total differential thermal stability resides in this multi-turn segment. Such a proportionality between DeltaDeltaG and incremental T(m) values is predicted for hybrid pairs exhibiting thermodynamic additivity in which the differential stability is predominantly enthalpic.
LeMaster
David M
DM
Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, University at Albany-SUNY, Empire State Plaza, Albany, NY 12201-0509, USA.
Tang
Jianzhong
J
Paredes
Diana I
DI
Hernández
Griselda
G
D000595:Amino Acid Sequence; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012416:Rubredoxins / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry
D010446:Peptide Fragments; D012416:Rubredoxins; D015032:Zinc