Two-dimensional correlation spectroscopy reveals coupled immunoglobulin regions of differential flexibility that influence stability.
Despite the well-accepted importance of protein flexibility and dynamics in molecular recognition and conformational stability, our understanding of these relationships is incomplete. Immunoglobulin flexibility is essential for antigen binding and adaptation to diverse molecular shapes and sizes. The inherent flexibility of immunoglobulins also renders these molecules suitable for investigating the possible relationships between protein flexibility and stability. To better understand these inter-relationships, we employ generalized perturbation-based two-dimensional correlation FTIR spectroscopy to monitor the time evolution of H-D exchange of an IgG1 as a function of pH. The differential flexibility of various immunoglobulin regions is described in response to an external perturbation and shown to vary widely. The greatest number of regions with differential exchange rates and, thus differential flexibility, is seen at pH 6. Approximately seven, six, five, and four separate states that exchange with different rates were observed at pH 6, 8, 4, and 2, respectively. The overall distribution of exchange rates calculated from the decays of the integrated Amide I and Amide II areas provides further evidence of multiple regions with differential flexibility. The sequence of events at pH 4 determined from the asynchronous vibrational patterns is of significant interest and suggests protonation of Glu and Asp side chains occurs first and initiates changes in the conformation and flexibility of different sheet and turns structure. A complex inter-relationship between differential regional flexibility and conformational coupling (i.e., cooperativity) initiated by changes in pH influences the stability of this IgG.
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA
Tim J
TJ
Kamerzell
C Russell
CR
Middaugh
D018360:Crystallography, X-Ray; D007136:Immunoglobulins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D018583:Pliability; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D007136:Immunoglobulins