The role of buried stack residues in the folding of the beta-helix domain of P22 tailspike

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 170-188).In-register, parallel alignment of similar or identical side chains is a common structural phenomenon in amyloid fibers. In the crystal structure of an amyloid fiber, these residues not only align, but take identical side chain orientations, creating long stacks of identical residues with identical orientations. This phenomenon of side chain stacking is a dominant component of the amyloid structure, and may take an equally important role in the seemingly sequence-independent formation of amyloid fibers or the sequence-dependent species barrier to amyloid transmission. Side chain stacking has long been observed as a prominent feature of the soluble parallel [beta]-helix fold, which exhibits a cross-[beta] structure reminiscent of amyloids. To investigate the sequence requirements of these stacks for directing a polypeptide chain into a [beta]-helical fold, we performed systematic mutational studies using the complete P22 tailspike protein, which contains a 13 rung [beta]-helix domain. The in vivo folding and assembly of 150 single mutant polypeptide chains were characterized at multiple temperatures by SDS-PAGE, which distinguishes the fully native, SDS-resistant state from partially folded or misfolded conformers. The vast majority of the buried core was completely intolerant to substitution at physiological temperatures, while only a few sites composing a continuously contacting network of residues called the folding spine were intolerant of alanine mutations at 18⁰C. These results indicate that the [beta]-helix folds through a processive folding mechanism, analogous to the nucleation and elongation mechanism of amyloids, but requiring recurring sequence-dependent stacking contacts that stretch the length of the [beta]-helix. Numerous avenues of investigation demonstrated that the observed folding defects of the full length tailspike were due to a sequence effect on the folding of the [beta]-helix domain.(cont.) Alanine mutants capable of reaching a native-like trimeric state were as thermostable as the wild-type trimer, indicating that mutants affect a folding intermediate rather than the native state. In vivo folding efficiencies of eight [beta]-helix folding mutants in the context of the isolated [beta]-helix domain paralleled the folding of the full length tailspike, demonstrating that these mutants act at the level of [beta]-helix folding. In vitro refolding of both full length and isolated [beta]-helix tailspike mutants demonstrated that the observed mutant folding deficiencies were solely caused by the single amino acid change and not due to interactions with cellular components. Folding spine mutations were further tested in the context of a suppressor mutation, A334V, which was previously shown to stabilize the [beta]-helix and rescue classic temperature sensitive folding mutations. Most mutations could not be rescued by the addition of A334V, a number of which were in fact further hindered in folding by the presence of the suppressor mutation. Eight folding spine mutations located between rungs 4 and 6 were rescued in a position specific manner, though only one of these positions directly contacts the suppressor. The pattern of rescue and hindrance observed with double mutants indicates that A334V is not a global suppressor and instead acts on a likely nucleus for folding. These studies provide evidence for a nucleation and elongation mechanism for folding of the parallel [beta]-helix. Single mutations throughout the [beta]-helix are capable of blocking this step-wise folding mechanism in a temperature dependent manner that likely leads to the population of a partially folded [beta]-helix species that would be inherently aggregation prone.by Ryan Matthew Simkovsky.Ph.D

Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus.

Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms

Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus.

A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells

Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus.

In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongates PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678-16683, 2012,http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide ofS. elongatus Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains

Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus.

In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongates PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678-16683, 2012,http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide ofS. elongatus Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains

Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus.

Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms

A microcin processing peptidase-like protein of the cyanobacterium Synechococcus elongatus is essential for secretion of biofilm-promoting proteins.

Small secreted compounds, e.g. microcins, are characterized by a double-glycine (GG) secretion motif that is cleaved off upon maturation. Genomic analysis suggests that small proteins that possess a GG motif are widespread in cyanobacteria; however, the roles of these proteins are largely unknown. Using a biofilm-proficient mutant of the cyanobacterium Synechococcus elongatus PCC 7942 in which the constitutive biofilm self-suppression mechanism is inactivated, we previously demonstrated that four small proteins, Enable biofilm formation with a GG motif (EbfG1-4), each with a GG motif, enable biofilm formation. Furthermore, a peptidase belonging to the C39 family, Peptidase transporter enabling Biofilm (PteB), is required for secretion of these proteins. Here, we show that the microcin processing peptidase-like protein encoded by gene Synpcc7942_1127 is also required for biofilm development - inactivation of this gene in the biofilm-proficient mutant abrogates biofilm development. Additionally, this peptidase-like protein (denoted EbfE - enables biofilm formation peptidase) is required for secretion of the EbfG biofilm-promoting small proteins. Given their protein-domain characteristics, we suggest that PteB and EbfE take part in a maturation-secretion system, with PteB being located to the cell membrane while EbfE is directed to the periplasmic space via its secretion signal

A microcin processing peptidase-like protein of the cyanobacterium Synechococcus elongatus is essential for secretion of biofilm-promoting proteins.

Small secreted compounds, e.g. microcins, are characterized by a double-glycine (GG) secretion motif that is cleaved off upon maturation. Genomic analysis suggests that small proteins that possess a GG motif are widespread in cyanobacteria; however, the roles of these proteins are largely unknown. Using a biofilm-proficient mutant of the cyanobacterium Synechococcus elongatus PCC 7942 in which the constitutive biofilm self-suppression mechanism is inactivated, we previously demonstrated that four small proteins, Enable biofilm formation with a GG motif (EbfG1-4), each with a GG motif, enable biofilm formation. Furthermore, a peptidase belonging to the C39 family, Peptidase transporter enabling Biofilm (PteB), is required for secretion of these proteins. Here, we show that the microcin processing peptidase-like protein encoded by gene Synpcc7942_1127 is also required for biofilm development - inactivation of this gene in the biofilm-proficient mutant abrogates biofilm development. Additionally, this peptidase-like protein (denoted EbfE - enables biofilm formation peptidase) is required for secretion of the EbfG biofilm-promoting small proteins. Given their protein-domain characteristics, we suggest that PteB and EbfE take part in a maturation-secretion system, with PteB being located to the cell membrane while EbfE is directed to the periplasmic space via its secretion signal