Measurement of protein interaction bioenergetics: application to structural variants of anti-sCD4 antibody.
This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.
Doyle
M L
ML
Department of Structural Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939, USA.
Brigham-Burke
M
M
Blackburn
M N
MN
Brooks
I S
IS
Smith
T M
TM
Newman
R
R
Reff
M
M
Stafford
W F
WF
Sweet
R W
RW
Truneh
A
A
Hensley
P
P
O'Shannessy
D J
DJ
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001666:Binding Sites, Antibody; D015704:CD4 Antigens / Q000737:chemistry* / Q000276:immunology*; D002151:Calorimetry / Q000379:methods; D002152:Calorimetry, Differential Scanning / Q000379:methods; D014644:Genetic Variation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008832:Microchemistry / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D020349:Surface Plasmon Resonance / Q000379:methods; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D046911:Macromolecular Substances