Local Folding and Misfoldingin the PBX Homeodomainfrom a Three-State Analysis of CPMG Relaxation Dispersion NMR Data.

NMR Carr–Purcell–Meiboom–Gill (CPMG)relaxationdispersion experiments represent a powerful approach for characterizingprotein internal motions and for gaining insight into fundamentalbiological processes such as protein folding, catalysis, and allostery.In most cases, CPMG data are analyzed assuming that the protein exchangesbetween two different conformational states. Systems exchanging amongmore than two states are far more challenging to characterize by CPMGNMR. For example, in the case of three-state exchange in the fasttime scale regime, it is difficult to uniquely connect the parametersextracted from CPMG analyses with the physical parameters of mostinterest, intercoversion rates, populations, and chemical shift differencesfor exchanging states. We have developed a grid search selection procedurethat allows these physical parameters to be uniquely determined fromCPMG data, based on additional information, which in this study comprisesligand-induced chemical shift perturbations. We applied this approachto the PBX homeodomain (PBX-HD), a three-helix protein with a C-terminalextension that folds into a fourth helix upon binding to DNA. We recentlyshowed that the C-terminal extension transiently folds, even in theabsence DNA, in a process that is likely tied to the cooperative bindingof PBX-HD to DNA and other homeodomains. Using the grid search selectionprocedure, we found that PBX-HD undergoes exchange between three differentconformational states, a major form in which the C-terminal extensionis unfolded, the previously identified state in which the C-terminalextension forms a fourth helix, and an additional state in which theC-terminal extension is misfolded. [ABSTRACT FROM AUTHOR]