Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor.
The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s(-1). For the mutants, dissociation rates were faster (0.022-0.025 s(-1)), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.
Svedhem
S
S
Division of Chemistry, Linköpings Universitet, Linköping, S-581 83, Sweden.
Enander
K
K
Karlsson
M
M
Sjöbom
H
H
Liedberg
B
B
Löfås
S
S
Mårtensson
L G
LG
Sjöstrand
S E
SE
Svensson
S
S
Carlsson
U
U
Lundström
I
I
D015374:Biosensing Techniques / Q000379:methods*; D024402:Carbonic Anhydrase II / Q000032:analysis* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D013449:Sulfonamides / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D000096926:Benzenesulfonamides
D024402:Carbonic Anhydrase II; D013449:Sulfonamides; D000096926:Benzenesulfonamides