TY - JOUR
T1 - Energetics of protein homodimerization
T2 - Effects of water sequestering on the formation of β-lactoglobulin dimer
AU - Bello, Martiniano
AU - Pérez-Hernández, Gerardo
AU - Fernández-Velasco, D. Alejandro
AU - Arreguín-Espinosa, Roberto
AU - García-Hernández, Enrique
PY - 2008/3
Y1 - 2008/3
N2 - Transient protein-protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein-protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine βlg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. ΔH and ΔCp were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near-UV CD spectra proved to be independent of protein concentration, indicating a rigid body-like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer's 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental ΔC p. Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces.
AB - Transient protein-protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein-protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine βlg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. ΔH and ΔCp were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near-UV CD spectra proved to be independent of protein concentration, indicating a rigid body-like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer's 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental ΔC p. Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces.
KW - Calorimetry
KW - ITC
KW - Protein-protein interaction
KW - Residual hydration
KW - Structural-energetics
UR - http://www.scopus.com/inward/record.url?scp=39749152155&partnerID=8YFLogxK
U2 - 10.1002/prot.21639
DO - 10.1002/prot.21639
M3 - Artículo
SN - 0887-3585
VL - 70
SP - 1475
EP - 1487
JO - Proteins: Structure, Function and Genetics
JF - Proteins: Structure, Function and Genetics
IS - 4
ER -