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-1) protein have generated conflicting results regarding whether HMG-1 exists as a monomer or is capable of oligomerizing to (functional) tetramers. To resolve this question, sedimentation velocity analysis yielded a s20,w value of 2.59S, which is consistent with a monomeric protein. Equilibrium sedimentation data were obtained for three HMG-1 concentrations at two rotor speeds. The six sets of data were fit to both an ideal single component and monomer-dimer equilibrium model, with essentially identical fits produced for both models, with the latter indicating a low extent (7%) of dimerization. Reaction of HMG-1 with glutaraldehyde produced a small population of oligomers consistent with a low level of dimers. This supported the monomer-dimer equilibrium model. Surprisingly, gel permeation chromatography yielded an apparent molecular mass of approx. 55 kDa for both HMG-1 and HMG-2. This finding is considered 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 HMG-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-linking studies strongly supports a moderately asymmetric monomer in solution and unequivocally eliminates the possibility of a highly extended shape for HMG-1 or the existence of any extensive oligomerization.
Ranatunga
W
W
Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403, USA.
Lebowitz
J
J
Axe
B
B
Pavlik
P
P
Kar
S R
SR
Scovell
W M
WM
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D005976:Glutaral; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D011487:Protein Conformation; D013950:Thymus Gland / Q000737:chemistry; D014867:Water / Q000737:chemistry
D003432:Cross-Linking Reagents; D006609:High Mobility Group Proteins; D014867:Water; D005976:Glutaral