Carboxyl pK(a) values and acid denaturation of BBL.
The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed (13)C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK(a) values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK(a) values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK(a), and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured (13)C(α) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.
Arbely
Eyal
E
MRC Centre for Protein Engineering, Hills Road, Cambridge, CB2 0QH, UK.
Rutherford
Trevor J
TJ
Neuweiler
Hannes
H
Sharpe
Timothy D
TD
Ferguson
Neil
N
Fersht
Alan R
AR
D002264:Carboxylic Acids / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
D002264:Carboxylic Acids; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D007655:Ketoglutarate Dehydrogenase Complex