NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin.
BACKGROUND
Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions.


RESULTS
Initial NMR studies indicated that the structures of the two complementary hybrids closely resemble "cut-and-paste" models derived from the parental Pf and Cp rubredoxins. This protein system offers a robust opportunity to characterize differences in solution structure, permitting the quantitative NMR chemical shift and NOE peak intensity data to be analyzed without recourse to the conventional conversion of experimental NOE peak intensities into distance restraints. The intensities for 1573 of the 1652 well-resolved NOE crosspeaks from the hybrid rubredoxins were statistically indistinguishable from the intensities of the corresponding parental crosspeaks, to within the baseplane noise level of these high sensitivity data sets. The differences in intensity for the remaining 79 NOE crosspeaks were directly ascribable to localized dynamical processes. Subsequent X-ray analysis of the metal binding site-swapped hybrids, to resolution limits of 0.79 A and 1.04 A, demonstrated that the backbone and sidechain heavy atoms in the NMR-derived structures lie within the range of structural variability exhibited among the individual molecules in the crystallographic asymmetric unit (approximately 0.3 A), indicating consistency with the "cut-and-paste" structuring of the hybrid rubredoxins in both crystal and solution.


CONCLUSIONS
Each of the significant energetic interactions in the metal binding site-swapped hybrids appears to exhibit a 1-to-1 correspondence with the interactions present in the corresponding parental rubredoxin structure, thus providing a structural basis for the observed additivity in conformational stability and dynamics. The congruence of these X-ray and NMR experimental data offers additional support for the interpretation that the conventional treatment of NOE distance restraints contributes substantially to the systematic differences that are commonly reported between NMR- and X-ray-derived protein structures.
Wadsworth Center, New York State Department of Health, School of Public Health, University at Albany - SUNY, Empire State Plaza, Albany, New York 12201, USA. lemaster@wadsworth.org
David M
DM
LeMaster
Janet S
JS
Anderson
Limin
L
Wang
Yi
Y
Guo
Hongmin
H
Li
Griselda
G
Hernández
D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D003013:Clostridium / Q000737:chemistry*; D018360:Crystallography, X-Ray; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D012416:Rubredoxins / Q000737:chemistry*; D012996:Solutions
D000577:Amides; D008670:Metals; D011994:Recombinant Proteins; D012416:Rubredoxins; D012996:Solutions; D008239:Lysine