Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.

Comparison of the biophysical characteristics of human brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor.

The structural properties of human brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were studied using sedimentation equilibrium and circular dichroism (CD), fluorescence and Fourier-transform infrared spectroscopies, and compared with those of human nerve growth factor (NGF). Both the far UV CD and infrared spectra indicate that these three proteins have similar, but not identical, secondary structures which contain primarily beta-sheet and irregular structures. NGF appears to contain the most beta-sheet while NT-3 contains a small fraction of alpha-helix. The near UV CD spectra appear to indicate that the three proteins contain disulfide bonds in similar environments, suggesting a resemblance in tertiary structure. The fluorescent tryptophans found in the molecules are relatively solvent exposed, while Trp102 found only in NT-3 is possibly quenched. The fluorescent Trp(s) in NGF are significantly quenched relative to those in the other two neurotrophic factors. Both NT-3 and BDNF have very hydrophilic surfaces at neutral pH, as indicated by a low binding affinity to a hydrophobic probe, anilinonaphthalenesulfonate. Sedimentation equilibrium showed that BDNF, NT-3, and NGF exist as strongly associated dimers in phosphate-buffered saline, pH 7.1. Fits of the observed fringe displacements to various association models suggested that the BDNF, NT-3, and NGF samples contain, in addition to the principal dimeric species, some oligomers, and that NT-3 contains a small fraction of incompetent monomer.

Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure.

We have recently described the identification, isolation, and characterization of a factor, termed stem cell factor (SCF), which acts on primitive hematopoietic progenitors of the marrow. A soluble form of the factor was isolated from the conditioned medium of a rat cell line (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201) and rat and human cDNAs have been cloned (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C.-H., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211). The cDNAs encode amino acids C-terminal to those found in the isolated natural form, including a putative transmembrane domain. This paper describes the structural characterization of soluble forms of recombinant human SCF purified from Escherichia coli (unglycosylated) and from Chinese hamster ovary (CHO) cells (glycosylated). Fluorescence emission spectra indicate that the single Trp residue is present in a hydrophobic environment. Circular dichroism and infrared spectroscopy indicate considerable secondary structure, including both alpha-helix and beta-sheet. Molecular weight determinations by sedimentation equilibrium show that the molecules are dimeric (noncovalently associated), and gel filtration analyses are consistent with this conclusion. The CHO cell-derived SCF is about 30% carbohydrate by weight, with both N-linked and O-linked sugar. The presence or absence of the carbohydrate does not influence the results of the various structural analyses.

Kinetic investigations of the quaternary enhancement effect and alpha/beta differences in binding the last oxygen to hemoglobin tetramers and dimers.

Analysis of O2 binding equilibria by two independent groups has suggested that the affinity for binding the fourth O2 to Hb tetramers is very high, about 800-1200 cal/mol higher than that of dimers (Chu, A. H., Turner, B. W., and Ackers, G. K. (1984) Biochemistry 23, 604-167; Di Cera, E., Robert, C. H., and Gill, S. J. (1987) Biochemistry 26, 4003-4008). Recently, Gibson and Edelstein challenged the reality of the quaternary enhancement effect, based on kinetic data (Gibson, Q. H., and Edelstein, S. J. (1987) J. Biol. Chem. 262, 516-519). However, these studies failed to directly address the key issue of the relative affinities of dimers and alpha 2 beta 2(O2)3. Furthermore, the extent to which alpha/beta differences influence these results remains an open question. Using partial laser photolysis and O2/CO replacement techniques we have, for the first time, resolved the rates of O2 association and dissociation to both alpha and beta chains within "R state" tetramers and dimers. We find that the beta chains are faster than alpha for both O2 binding (approximately 2-fold) and release (approximately 3-fold). The kinetically determined O2 affinities derived from these data are essentially identical for dimers and alpha 2 beta 2(O2)3. That is, the data do not show significant quaternary enhancement and suggest that the equilibrium data have both overestimated the affinity of alpha 2 beta 2(O2)3 and underestimated the affinity of dimers. The significance of and possible origins for the discrepancy between equilibrium and kinetic data are discussed.

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly.

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.