The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae

208 circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of ureainduced unfolding. Temperature and urea denaturation experiments show that Tm 143-235 is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of ␤-branched residues located at d positions.The coiled-coil motif mediates the process of oligomerization of many proteins. This structure is a consequence of a heptapeptide repetition (abcdefg) in the chemical nature of the residues in the primary structure of the polypeptide chain (1-3). An ␣-helical conformation places hydrophobic residues at positions a and d on the same side of the helix, creating a nonpolar interface that promotes dimerization through the burial of hydrophobic surface. Residues at positions e and g are often charged and may form salt bridges with residues at positions g and e, respectively, of the other helix. The maximization of favorable ionic attractions and the minimization of unfavorable repulsions probably influence the particular alignment of the ␣-helical chains (4). The folding of synthetic peptides that adopt a coiled-coil structure has been investigated extensively (4 -11). Synthetic peptide models for coiled-coils may not always be taken as representative because they often employ regular patterns of residues at positions a and d and lack i, iϩ3 and i, iϩ4 intrahelical ionic interactions (7,12). Natural coiled-coil sequences, such as tropomyosin (Tm), 1 are more complex, with irregular patterns of intrahelix and interhelix side chain-side chain interactions and a greater variety of residues at the hydrophobic core. Tm can be used as a natural model for the study of the principal interactions that maintain the coiled-coil structure and ␣-helix stability.Muscle ␣-Tm is a symmetric coiled-coil composed of two parallel and in register ϳ410 Å, 284-residue ␣-helices (2, 13, 14). The C-terminal half of the Tm molecule is less thermally stable than the N-terminal half (15,16). A series of studies (17,18) have suggested that Tm fragments with less than 94 residues were unable to form stable secondary structures at low micromolar concentrations. Recently, Holtzer et al. (19) showed that a 65-amino acid fragment (residues 190 -254) presents a significant amount of ␣-helix (ϳ43%) when present in high micromolar concentrations (115 M) at 10°C but is essentially unfolded at 25°C.Microcalorimetric analysis of ␣-Tm thermal denaturation has been interpreted as a multistep process in which spec...

The members of the ezrin-radixin-moesin (ERM) family of proteins function as membranecytoskeletal cross-linkers in actin-rich cell surface structures. ERM proteins are thereby thought to be essential for cortical cytoskeleton organization, cell motility, adhesion, and proliferation. These modular polypeptides consist of a central helix-rich region, termed the R-domain, that connects an N-terminal FERM domain required for membrane binding and a C-terminal region which contains a major actinbinding motif. Conformational regulation of ERM protein function occurs by association of the FERM and C-terminal domains, whereby the membrane-and actin-binding activities are mutually suppressed and the protein is thought to take an inactive "closed" form. Here we report in Vitro and in ViVo studies of radixin to address the role of the R-domain in conformational activation of ERM proteins. Remarkably, an isolated R-domain comprised of radixin 311-469 forms a monomeric, stable helical rod that spans 240 Å in length from the N-terminus to the C-terminus, most likely stabilized by extensive salt bridge interactions. By fusing green fluorescent protein variants to the FERM and C-terminal domains, we probed in Vitro conformational changes impacted by the presence of the R-domain using fluorescence resonance energy transfer (FRET). Furthermore, deletion of this unusually long R-helical structure (radixin residues 314-411) prevents ERM membrane targeting in ViVo.

The amphipathic alpha-helices of exchangeable apolipoproteins (apo) function to simultaneously facilitate interaction with lipid surfaces and the aqueous environment. In contrast to mammalian apoA-I's, which self-associate in the absence of lipid, chicken apoA-I, which shares 66% sequence homology with human apoA-I, exists as a monomeric protein when dissociated from high-density lipoprotein (HDL). Sedimentation equilibrium studies conducted in the analytical ultracentrifuge yielded a weight-average molecular weight of 28,170. Corresponding sedimentation velocity and diffusion experiments gave rise to s0(20,w) = 2.23 S and D0(20,w) = 6.39 x 10(-7) cm2/s. A translational frictional ratio (f/fmin) of 1.18 and an axial ratio of 4.0 were also determined from this data. The Stokes radius (Rs,sed = 2.80 nm) and translational frictional ratio were subsequently used to calculate estimated molecular dimensions of 25.2 x 100.8 A for chicken apoA-I. Circular dichroism (CD) studies revealed a highly alpha-helical structure predicted to be 74% by Provencher-Glöckner analysis. Denaturation studies performed on lipid-free apoA-I and monitored by CD revealed a midpoint of denaturation of 0.64 M guanidine hydrochloride. From plots of delta G(app) versus guanidine hydrochloride concentration, a delta GDH2O of 1.86 kcal/mol was determined. In other studies, a midpoint of temperature-induced denaturation for apoA-I of 57 degrees C was obtained. The effect of solvent pH on the secondary structure content of apoA-I revealed a significant loss of alpha-helix below pH 4.0 and above pH 10, suggesting that lipid-free apoA-I may by partially stabilized by the formation of intra- or interhelix salt bridges between oppositely charged amino acid side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzyme CoA transferase from porcine heart (EC 2.8.3.5) is a homodimer; each subunit consists of two domains linked by a hydrophilic "hinge" region. We have prepared separate DNA segments encoding each of these domains. Incorporation of these two DNA segments within an operon or within two separate transcription units does not preclude the synthesis and assembly of CoA transferase in Escherichia coli. When the two domain fragments are produced and purified individually from separate cultures and subsequently mixed, enzyme activity accumulates to near wild-type levels only after a lengthy incubation. Each domain is more susceptible to aggregation than wild-type CoA transferase. Circular dichroism shows that, prior to mixing, the domains possess a different secondary structural profile compared to their counterparts in the native enzyme. However, mixing and incubation of the domains produces a complex with far-UV CD, fluorescence, and ultracentrifugation properties similar to those of wild-type CoA transferase. Finally, we show that the intact hydrophilic peptide which links the two domains is essential for the recovery of activity observed upon refolding of the denatured enzyme in vitro. These results indicate that the folding and assembly of pig heart CoA transferase require a productive interaction between its two domains, involving a substantial conformational rearrangement.