Thermal stability of calmodulin and mutants studied by (1)H-(15)N HSQC NMR measurements of selectively labeled [(15)N]Ile proteins.
Calmodulin, the Ca(2+)-dependent activator of many cellular processes, contains two well-defined structural domains, each of which binds two Ca(2+) ions. In its Ca(2+)-free (apo) form, it provides an attractive model for studying mechanisms of protein unfolding, exhibiting two separable, reversible processes, indicating two structurally autonomous folding units. (1)H-(15)N HSQC NMR in principle offers a detailed picture of the behavior of individual residues during protein unfolding transitions, but is limited by the lack of dispersion of resonances in the unfolded state. In this work, we have used selective [(15)N]Ile labeling of four distinctive positions in each calmodulin domain to monitor the relative thermal stability of the folding units in wild-type apocalmodulin and in mutants in which either the N- or C-domain is destabilized. These mutations lead to a characteristic perturbation of the stability (T(m)) of the nonmutated domain relative to that of wild-type apocalmodulin. The ability to monitor specific (15)N-labeled residues, well-distributed throughout the domain, provides strong evidence for the autonomy of a given folding unit, as well as providing accurate measurements of the unfolding parameters T(m) and DeltaH(m). The thermodynamic parameters are interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin, where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changes in the observed unfolding behavior of a given folding unit. The selective (15)N labeling approach is thus a general method that can provide detailed information about structural intermediates populated in complex protein unfolding processes.
Biekofsky
Rodolfo R
RR
Division of Molecular Structure and Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K.
Martin
Stephen R
SR
McCormick
John E
JE
Masino
Laura
L
Fefeu
Sandrine
S
Bayley
Peter M
PM
Feeney
James
J
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D004331:Drosophila melanogaster / Q000737:chemistry; D007532:Isoleucine / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D002147:Calmodulin; D009587:Nitrogen Isotopes; D007532:Isoleucine; D002118:Calcium