Reexamination of the high mobility group-1 protein for self-association and characterization of hydrodynamic properties

Previous studies of the 25 kDa high mobility group-1 (HMG-I) protein have g enerated conflicting results regarding whether HMG-I exists as a monomer or is capable of oligomerizing to (functional) tetramers. To resolve this que stion, sedimentation velocity analysis yielded a s(20,w) value of 2.59S, wh ich is consistent with a monomeric protein. Equilibrium sedimentation data were obtained for three HMG-1 concentrations at two rotor speeds. The six s ets of data were fit to both an ideal single component and monomer-dimer eq uilibrium model, with essentially identical fits produced for both models, with the latter indicating a low extent (7%) of dimerization. Reaction of H MG-1 with glutaraldehyde produced a small population of oligomers consisten t with a low level of dimers. This supported the monomer-dimer equilibrium model. Surprisingly, gel permeation chromatography yielded an apparent mole cular mass of approx. 55 kDa for both HMG-1 and HMG-2. This finding is cons idered anomalous and presumably due to the high negative charge density in the C terminus of HMG-1. The sedimentation data also permit one to model HM G-1 as a hydrated prolate ellipsoid with a major axis/minor axis ratio of 2 .79. The collective evidence from the sedimentation and chemical cross-link ing studies strongly supports a moderately asymmetric monomer in solution a nd unequivocally eliminates the possibility of a highly extended shape for HMG-1 or the existence of any extensive oligomerization. (C) 1999 Elsevier Science B.V. All rights reserved.