Cooperative hydrogen bond interactions in the streptavidin–biotin system

The thermodynamic and structural cooperativity between the Ser45– and D128–biotin hydrogen bonds was measured by calorimetric and X‐ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity ∼2 × 107 times lower than that of wild‐type...

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Bibliographic Details
Published in:Protein science 2006-03, Vol.15 (3), p.459-467
Main Authors: Hyre, David E., Le Trong, Isolde, Merritt, Ethan A., Eccleston, John F., Green, N. Michael, Stenkamp, Ronald E., Stayton, Patrick S.
Format: Article
Language:English
Subjects:
45/128 S45A/D128A, double mutant of streptavidin
Biotin - chemistry
Calorimetry
cooperativity
Crystallography, X-Ray
hydrodynamics
hydrogen bond
Hydrogen Bonding
ITC, isothermal calorimetry
Kinetics
MD, molecular dynamics
Models, Molecular
molecular recognition
Mutation
Protein Binding
SPR, surface plasmon resonance
StAv, streptavidin
streptavidin
Streptavidin - chemistry
Streptavidin - genetics
structure
Thermodynamics
X‐ray crystallography
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Summary:The thermodynamic and structural cooperativity between the Ser45– and D128–biotin hydrogen bonds was measured by calorimetric and X‐ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity ∼2 × 107 times lower than that of wild‐type streptavidin at 25°C. The corresponding reduction in binding free energy (ΔΔG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11‐fold greater than that predicted by a linear combination of the single‐mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128–biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild‐type hydrogen bond between D128–OD and biotin–N2 is thermodynamically stronger than that between S45–OG and biotin–N1.
ISSN:0961-8368
1469-896X
DOI:10.1110/ps.051970306