Protein Stability, Folding, Macromolecular Associations

Heart, Lung, And Blood Institute

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Abstract

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. 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 E+6 1/M . 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, enzyme I. Accordingly, intracellular concentrations of Mg(II), PEP, and pyruvate together control the activity of enzyme I. Future plans (involving EF, AG, and AP) include work on an important new nitrogen signal pathway of E. coli, which involves phosphorylation of a His residue in the first enzyme by phosphoenolpyruvate. These studies are in collaboration with G. Wang (University of Nebraska Medical Center) who is determining NMR structures and Alan Peterkofsky (NHLBI) who is expressing proteins cloned by Yeong-Jae Seok (Dir., Microbiology, Seoul National University) who is working during the Summer months in Alan Peterkofsky?s laboratory. We will be conducting biophysical and thermodynamic measurements. Cardiac-specific Csx/Nkx2.5 homeodomain - protein stability and energetics of DNA interactions:(EF, AG, JMG, JWM, J-SM, J-H J JAF) The mouse cardiac-specific Nkx2.5 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 Nkx2.5 homeodomain since preliminary studies showed that Cys56 caused problems both in the isolation and subsequent thermal unfolding studies. NMR studies have shown that the structures of Nkx2.5 and Nkx2.5(C56S) are the same as the vnd/NK-2 when bound to the same specific DNA. Thermal unfolding of Nkx2.5(C56S) at both pH 6.0 and 7.4 in the absence and presence of NaCl is a reversible, two-state process with unit cooperativity, as measured by DSC and CD. Titrations of specific 18 bp DNA with Nkx2.5(C56S) in buffer at pH 7.4 with 100 mM NaCl yield binding constants of 2 - 6 E+8 from 10-37 C and a stoichiometry of 1:1, using ITC. DNA binding by Nkx2.5(C56S) is enthalpically controlled with a heat capacity change of -0.19 kcal K-1mol-1, which gives a good estimate of the buried apolar surface area on complex formation (648 +/- 36 square Angstroms). Intrinsic Trp fluorescence changes upon urea-induced 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. The latter studies also revealed that the vnd/NK-2 HD is much more flexible in the free form which can partially explain why thermodynamic parameters differ for Nkx2.5(C56S) and NK-2 binding specifc DNA. The work of Dr. Fodor is particularly important because mutations in the human NKX2.5 homeodomain (identical in sequence to the mouse Nkx2.5 HD) have been found to produce abnormalities in human embryonic heart development and, in the future, the specific defects of such mutants will be probed. Human mitochondrial ClpP protease and ClpX chaperone interaction: (MND, AG, MRM, SGK) The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (7.0 S; 169,200 Mr) at concentrations >3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by sedimentation equilibrium measurements has an apparent molecular weight of 1,000,000. Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles. Brain myosin Va tail fragment-light chain (LC8b) interaction: (EF, AG, JAH, WW) The interaction of a brain myosin V tail fragment (myoVa, 28.6 kDa dimer) with light chain (LC8b, 20.8 kDa dimer) is being studied by far-UV circular dichroism (CD), calorimetric (DSC), and analytical ultracentrifugal techniques. The binding of LC8b to the wild-type myoVa fragment containing the Exon B motif has been found to produce an increase in the unfolding transition temperature of the coiled coil of myoVa, whereas LC8b has no effect on a deletion mutant of the myosin Va tail fragment missing the DDK sequence of Exon B. Also, thermal unfolding of myoVa is a reversible two-state process in CD, but exhibits two domains with differing thermal stability and low cooperativity in DSC. [The results from these studies will be presented by WW and JAH at the Cell Biology meetings in Dec. 2005.]