The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(II)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(II)) promotes essentially complete dimerization of EI(H189A) at pH 7.5 and 20 C, 5 mM pyruvate (with 2 mM Mg(II)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(II) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from log K >8 to 5.7 or 7.4 (per M monomer), respectively. With 2 mM Mg(II) at 15-25 C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with an apparent association constant of 1.0 E6 1/M (DeltaG = -8.05 ? 0.05 kcal/mol) and DeltaH = +3.9 kcal/mol at 20 C with a heat capacity change of -0.33 kcal/(Kmol). The binding of PEP to EI(H189A) is synergistic with that of Mg(II). Thus, physiological concentrations of PEP and Mg(II) increase, whereas pyruvate and Mg(II) decrease, the amount of dimeric, active, dephospho-enzyme I. Accordingly, intracellular concentrations of Mg(II), PEP, and pyruvate together control the activity of Enzyme I. Myosin I heavy chain tail (48,645 kDa) from Acanthamoeba: (EF, AG, EK, FM) Sedimentation velocity studies have shown that this protein is monomeric at pH 7.5 in low salt at 1.7 mg/mL concentration. The observed aggregation at higher concentrations of the myosin I tail is being investigated. Human NKx-2.5 homeodomain protein stability and DNA interactions:(EF, AG, JWM, J-SM, JAF) The NKx-2.5 homeodomain (residues 1-79; Mr 9691) is 73% identical with the parent vnd/NK-2 homeodomain protein from Drosophila melanogaster previously studied in our laboratory by Gonzalez et al. (Biochemistry 40, 4923-4931, 2001). We have used the C56S mutant of the wild-type NKx-2.5 homeodomain since preliminary studies showed that Cys56 caused problems both in the isolation and subsequent thermal unfolding studies. Also, NMR experiments show that the structure of NKx-2.5 is not perturbed by replacing Cys with Ser. Thermal unfolding of NKx-2.5 at both pH 6.0 and 7.4 in the absence and presence of NaCl is fully reversible as measured by DSC and CD. ITC measurements to determine the thermodynamics of the interaction of NKx-2.5(C56S) with specific 18 bp DNA also have been performed. Intrinsic Trp fluorescence changes upon thermal unfolding of NKx-2.5(C56S) have shown that the single, conserved Trp 48 in helix III is severely quenched in the folded, native state, as it is in other homeodomain proteins. CARMIL from Acanthamoeba and heterodimer Capping Protein Interaction: (MND, AG, KR, JAH) Purified CARMIL (subunit Mr = 121,610) preparations in the absence of Capping Protein are homogeneous as evidenced in sedimentation velocity experiments which show a single symmetrical boundary. At 9.4 micromolar subunit, CARMIL has a sedimentation coefficient of 7.1 S, under which conditions the protein is >90% dimer. This indicates that the CARMIL dimer is quite asymmetric with a frictional coefficient of ca. 2.0 or about 2-fold greater than that of a spherical particle. Global fitting of sedimentation equilibrium data at 4 C obtained at 34 and 36 h at 7500 rpm for two CARMIL preparations gave excellent fits to a monomer - dimer equilibrium model with an association constant (log K) of 6.0 /(M monomer) at pH 7.0. No higher oligomeric species thaan dddimer could be detected. Apparently, the dimerization constant is greater at 20 C since only dimer is detected in sedimentation velocity time-derivative profiles for 4-7 micromolar CARMIL subunit at the higher temperature. Once CARMIL and the Capping heterodimer protein (CP) had been separately purified and the extinction coefficients determined, these proteins were mixed in a known ratio to give 1.7-fold CP:CARMIL for a sedimentation equilibrium experiment. After reaching equilibrium, the data have been analyzed (assuming three models) for the different components present. Only one model was consistent with the fact that CARMIL monomer could not be detected and that the presence of CP promotes dimerization of CARMIL. For the mixture, interference data are fitted well to a model with free CP (calculated from [3.28 micromolar total - complexed CP]), 13-15% CARMIL dimer, and 85-87% CARMIL dimer complexed to one equivalent of CP. Moreover, the calculated association constant, log K = 6.4 (/M) for CP binding to CARMIL dimer from the sedimentation equilibrium data is in agreement with the value measured for the affinity of CP for CARMIL in a BioCore assay. The fact that only one equivalent of CP interacts with the CARMIL dimer suggests that there is severe negative cooperativity in binding the second equivalent of CP or, as is more likely, the dimerization of CARMIL blocks a second binding site for CP. Human ClpP Protease and ClpX Chaperone Interaction: (MND, AG, MRM, SGK) In both sedimentation velocity and sedimentation equilibrium experiments, purified human ClpP (hClpP) is a monodisperse heptamer of 24,166 Mr subunits (7.0 S). This result is surprising since in E. coli, the two 7-membered rings of ClpP are assembled under native conditions into a stable tetradecamer without free heptamer present. Since in the mitochondria hClpP is complexed to hClpX, an approach to equilibrium at low speeds has been used with absorption optics to examine mixtures of hexameric hClpX with heptameric hClpP (0.25, 0.50, and 1.0 equiv of hexameric ClpX to 14-mer ClpP in the presence of ATPgammaS. A 2:1 complex between hexameric hClpX and tetradecameric hClpP accounted for ca. 35% of the ClpP present when 1.0 equiv of hexameric ClpX to 14-mer ClpP was present (with ca. 8% free heptameric ClpP). Other possible subassemblies (such as 14-mer ClpP, heptameric ClpP complexed to hexameric ClpX, or 14-mer ClpP complexed to one equiv of hexameric ClpX ) are not present in sufficient quantities to be detected. Thus, the interaction of hexameric ClpX with heptameric ClpP strongly stabilizes the tetradecameric structure of ClpP in a complex flanked by hexameric rings of ClpX (analogous to the E. coli ClpAP complex). This experiment has been repeated with a 1:1 mixture of heptameric ClpP and hexameric ClpX in the presence of ATPgS using interference optics at 3500 rpm and 12 C. Again, the only significant species present after 6 days is the complex: 2 ClpX hexamers: tetradecameric ClpP. This means that once this complex is formed, it is thermodynamically stable and does not require nucleotide present since ATPgammaS would by hydrolyzed after 6 days at 12 C. Cel 45 mutant (cellulase) and Cel 45 core: (MND,AG, GR) We also were approached by G. Rialdi to run sedimentation velocity experiments at both pH 4.2 and pH 7.8 of an inactive mutant D10N of Cel-45 core (containing no carbohydrate) with Mr 22,865 and Cel-45 wild-type cellulase, containing 8-12 k Da carbohydrate (Mr 40,200 ). Cel-45 appeared to form a dimer at the higher pH in isoelectric electrophoresis studies. As anticipated, the core Cel-45 was monomeric at both acid and neutral pH. However, the wild-type Cel-45 in sedimentation velocity runs also is monomeric at both pH?s. Prof. Rialdi now is investigating whether Cel-45 interacts with ampholytes in gels at the higher pH.