Developmental regulation of the cell division protein FtsZ in Anabaena sp. strain PCC 7120, a cyanobacterium capable of terminal differentiation

Heterocysts are terminally differentiated cells devoted to nitrogen fixation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. We show here that the cell division protein FtsZ is present in vegetative cells but undetectable in heterocysts. These results provide a first rational explanation for the inability of mature heterocysts to undergo cell division.     

Side chain accessibility and dynamics in the molten globule state of alpha-lactalbumin: a (19)F-NMR study

The molten globule state of alpha-lactalbumin (alpha-LA) has been considered a prototype of partially folded proteins. Despite the importance of molten globules in understanding the mechanisms of protein folding and its relevance to some biological phenomena, site-specific information on the structure and dynamics of a molten globule is limited, largely because of the high conformational flexibility and heterogeneity. Here, we use selective isotope labeling and (19)F NMR to investigate the solvent accessibility and side-chain dynamics of aromatic residues in the molten globule of alpha-LA. Comparison of these properties with those of the native and unfolded protein indicates that the alpha-LA molten globule is highly heterogeneous; each residue has its unique solvent accessibility and motional environment. Many aromatic residues normally buried in the interior of native alpha-LA remain significantly buried in the molten globule and the side-chain dynamics of these residues are highly restricted. Our results suggest that hydrophobic and van der Waals interactions mediated by the inaccessible surface area could be sufficient to account for all the stability of the alpha-LA molten globule, which is approximately 50% of the value for the native protein.     

Characterization of the functionally related sites in the neural inducing gene noggin

Previously we have shown that blocking bone morphogenetic protein (BMP) receptor signaling by a dominant negative BMP receptor causes neurogenesis in Xenopus animal caps (ACs), whereas the physiological neural inducer noggin acts as a homodimer physically binding to BMP-4 and disrupting its signaling at the ligand level. The present study attempted to elucidate the relationship between the structure and function of noggin. By replacing some cysteine residues with serine residues through a site-directed mutagenesis strategy, we generated three noggin mutants, C145S, C205S, and C(218, 220, 222)S (3CS). Although mRNAs encoded by these mutants were translated as efficiently as wild-type (WT) noggin mRNA, they behaved differently when expressed in vivo. Expression of WT noggin or C205S in Xenopus ACs converted the explants (prospective ectoderm) into neural tissue, indicated by the neural-like morphology and expression of the pan neural marker NCAM in the ACs. In contrast, ACs expressing C145S or 3CS sustained an epidermal fate like the control caps. Similar results were observed in the mesoderm where C205S (but not C145S and 3CS) displayed dorsalizing activity as well as WT noggin. Altogether, our results suggest that Cys145 alone or Cys(218, 220, 222) as a whole in noggin protein is required for the biological activities of noggin, probably participating in the dimerization of noggin with BMP-4 or itself.     

Evidence that beta-tubulin induces a conformation change in the cytosolic chaperonin which stabilizes binding: implications for the mechanism of action

The class II chaperonin CCT facilitates protein folding by a process that is not well-understood. One striking feature of this chaperonin is its apparent selectivity in vivo, folding only actin, tubulin, and several other proteins. In contrast, the class I chaperonin GroEL is thought to facilitate the folding of many proteins within Escherichia coli. It has been proposed that this apparent selectivity is associated with certain regions of a substrate protein's primary structure. Using limiting amounts of beta-tubulin, beta-tubulin mutants, and beta-tubulin/ftsZ chimeras, we assessed the contribution of select regions of beta-tubulin to CCT binding. In a complementary study, we investigated inter-ring communication in CCT where we exploited polypeptide binding sensitivity to nucleotide to quantitate nucleotide binding. beta-Tubulin bound with a high apparent affinity to CCT in the absence of nucleotide (apparent K(D) approximately 3 nM; its apparent binding free energy, DeltaG, ca. -11.8 kcal/mol). Despite this, the interactions appear to be weak and distributed throughout much of the sequence, although certain sites ("hot spots") may interact somewhat more strongly with CCT. Globally averaged over the beta-tubulin sequence, these interactions appear to contribute ca. -9 to -11 cal/mol per residue, and to account for no more than 50-60% of the total binding free energy. We propose that a conformation change or deformation induced in CCT by substrate binding provides the missing free energy which stabilizes the binary complex. We suggest that by coupling CCT deformation with polypeptide binding, CCT avoids the need for high "intrinsic" affinities for its substrates. This strategy allows for dynamic interactions between chaperonin and bound substrate, which may facilitate folding on the interior surface of CCT in the absence of nucleotide and/or productive release of bound polypeptide into the central cavity upon subsequent MgATP binding. CCT displayed negative inter-ring cooperativity like GroEL. When ring 1 of CCT bound MgATP or beta-tubulin, the affinity of ring 2 for polypeptide or nucleotide was apparently reduced approximately 100-fold.     

Evidence for overlapping and distinct functions in protein transport of coat protein Sec24p family members

Budding of transport vesicles from the endoplasmic reticulum in yeast requires the formation, at the budding site, of a coat protein complex (COPII) that consists of two heterodimeric subcomplexes (Sec23p/Sec24p and Sec13p/Sec31p) and the Sar1 GTPase. Sec24p is an essential protein and involved in cargo selection. In addition to Sec24p, the yeast Saccharomyces cerevisiae expresses two non-essential Sec24p-related proteins, termed Sfb2p (product of YNL049c) and Sfb3p/Lst1p (product of YHR098c). We here show that Sfb2p and, less efficiently, Sfb3p/Lst1p are able to bind, like Sec24p, the integral membrane cargo protein Sed5p. We also demonstrate that Sfb2p, like Sec24p and Sfb3p/Lst1p, forms a complex with Sec23p in vivo. Whereas the deletion of SFB2 did not affect transport kinetics of various proteins, the maturation of the glycolipid-anchored plasma membrane protein Gas1p was differentially impaired in sfb3 knock-out cells. We generated several conditional-lethal sec24 mutants that, combined with null alleles of SFB2 and SFB3/LST1, led to a complete block of transport between the endoplasmic reticulum and the Golgi (sec24-11/Deltasfb2) or to cell death (sec24-11/Deltasfb3). Of the Sec24p family members, Sfb2p is the least abundant at steady state, but high intracellular concentrations of Sfb2p can rescue sec24 mutants under restrictive conditions. The data presented strongly suggest that the Sec24p-related proteins function as COPII components.     

Specificity determinants for the pyruvate dehydrogenase component reaction mapped with mutated and prosthetic group modified lipoyl domains

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys(173)), with three analogs substituted for the lipoyl group on Lys(173), and with selected L1 mutants. Besides Lys(173) mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 A on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. Alignment of over 40 lipoyl domain sequences supports Ser(141), Thr(143), and Glu(179) serving as specificity residues for use by E1 from eukaryotic sources. Extensive interactions of the lipoyl-lysine prosthetic group within the active site are supported by the limited inhibition of E1 acetylation of native L2 by L2 domains altered either by mutation of Lys(173) or enzymatic addition of lipoate analogs to Lys(173). Thus, efficient use by mammalian E1 of cognate lipoyl domains derives from unique surface residues with critical interactions contributed by the universal lipoyl-lysine prosthetic group, key specificity residues, and some conserved residues, particularly Asp(172) adjacent to Lys(173).