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Samantha Miller

Publications and source records attributed to Samantha Miller.

9 recordsLinked to original sources
PubMed2005

Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel.

The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was recently solved. However, the conformation of the closed state and the gating transition remain uncharacterized. The pore-lining transmembrane helix contains a conserved glycine- and alanine-rich motif that forms a helix-helix interface. We show that introducing 'knobs' on the smooth glycine face by replacing glycine with alanine, and substituting conserved alanines with larger residues, increases the pressure required for gating. Creation of a glycine-glycine interface lowers activation pressure. The importance of residues Gly104, Ala106 and Gly108, which flank the hydrophobic seal, is demonstrated. A new structural model is proposed for the closed-to-open transition that involves rotation and tilt of the pore-lining helices. Introduction of glycine at Ala106 validated this model by acting as a powerful suppressor of defects seen with mutations at Gly104 and Gly108.

Alanine↗
PubMed2004

Gating the bacterial mechanosensitive channels: MscS a new paradigm?

Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the millisecond timescale they must achieve the transition from tightly closed oligomers to large, relatively non-discriminating pores. The crystal structure for MscL, combined with genetic and biochemical analysis, provided the initial insights for the mechanism by which this structural transition might be made. Discovery of the gene for a second class of mechanosensitive channel, MscS, and its subsequent crystallisation, has provided a new paradigm for mechanosensation, enabling a deeper understanding of the mechanisms of sensing membrane tension.

Escherichia coli↗
PubMed2003

A role for mechanosensitive channels in survival of stationary phase: regulation of channel expression by RpoS.

The mechanosensitive (MS) channels MscS and MscL are essential for the survival of hypoosmotic shock by Escherichia coli cells. We demonstrate that MscS and MscL are induced by osmotic stress and by entry into stationary phase. Reduced levels of MS proteins and reduced expression of mscL- and mscS-LacZ fusions in an rpoS mutant strain suggested that the RNA polymerase holoenzyme containing sigmaS is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of sigmaS is direct, the MscS and MscL promoters both use RNA polymerase containing sigmaS in vitro. Conversely, clpP or rssB mutations, which cause enhanced levels of sigmaS, show increased MS channel protein synthesis. RpoS null mutants are sensitive to hypoosmotic shock upon entry into stationary phase. These data suggest that MscS and MscL are components of the RpoS regulon and play an important role in ensuring structural integrity in stationary phase bacteria.

Bacterial Proteins↗
PubMed2003

The closed structure of the MscS mechanosensitive channel. Cross-linking of single cysteine mutants.

Mechanosensitive channels must make a large conformational change during the transition from the closed to the open state. The crystal structure of the open form of the Escherichia coli MscS channel was recently solved and depicts a homoheptamer (1). In this study, cross-linking of site-specific cysteine substitutions demonstrates that residues up to 10-33 A apart in the crystal structure readily form disulfide bridges in the closed form and can also be cross-linked by a 10-A linker. Cross-linking between adjacent subunits stabilizes the heptameric form of the channel providing biochemical evidence to support the crystal structure. The data are consistent with the published model (1) in that the membrane domain is highly flexible and that the closed to open transition may involve a significant displacement of transmembrane helices 1 and 2, possibly by as much as 30 A. The data are also consistent with significant flexibility of the cytoplasmic domain.

Base Sequence↗
PubMed2003

Domain organization of the MscS mechanosensitive channel of Escherichia coli.

The major structural features of the Escherichia coli MscS mechanosensitive channel protein have been explored using alkaline phosphatase (PhoA) fusions, precise deletions and site-directed mutations. PhoA protein fusion data, combined with the positive-inside rule, strongly support a model in which MscS crosses the membrane three times, adopting an N(out)-C(in) configuration. Deletion data suggest that the C-terminal domain of the protein is essential for the stability of the MscS channel, whereas the protein will tolerate small deletions at the N-terminus. Four mutants that exhibit either gain-of-function (GOF) or loss-of-function have been identified: a double mutation I48D/S49P inactivates MscS, whereas the MscS mutants T93R, A102P and L109S cause a strong GOF phenotype. The similarity of MscS to the last two domains of MscK (formerly KefA) is reinforced by the demonstration that expression of a truncated MscK protein can substitute for MscL and MscS in downshock survival assays. The data derived from studies of the organization, conservation and the influence of mutations provide significant insights into the structure of the MscS channel.

Alkaline Phosphatase↗
PubMed2003

Risk and protective factors for juvenile eating disorders.

Eating disorders are prevalent and complicated disorders which are difficult to treat. Unicausal and main effects models are not likely to do justice to the complexity of psychopathology encountered, as one considers etiology and pathogenesis. Risk and protection can arise out of several domains: biological, psychological and social. Risk and protective factors aggregate in specific developmental phases and interact to produce adverse outcomes. Temperamental factors, eating dysregulation, attachment, deficient self regulation and sociocultural ideals of health and beauty all contribute to pathogenesis. Applying the insights of developmental psychopathology to these disorders has considerable potential to lead to early and preventive interventions. Reviewing the current literature from this perspective and updating a similar discussion from 8 years ago, we witness a continued accumulation of quality empirical data. Compared to previous reviews, the field's attention has shifted to psychosocial/cultural domains relevant to eating, away from biological risk. In the aggregate, these data make possible the increasing differentiation of eating disorders from other psychopathology, and the specific pathways in which anorexia and bulimia may develop. Understanding of risk and vulnerability still outweighs our knowledge of protective factors and resilience. While an ideal study would be longitudinal, such studies are still extremely difficult to conduct and costly, thus, forcing us to further our understanding from lagged designs, cross-sectional data and case control studies. While these have many limitations, they do seem to produce an increasingly coherent account of the development of these disorders and prepare us for more targeted and longitudinal study of high risk populations.

Adolescent↗
PubMed2003

Two families of mechanosensitive channel proteins.

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.

Amino Acid Sequence↗
PubMed2002

A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch.

The regulation of cation content is critical for cell growth. However, the molecular mechanisms that gate the systems that control K+ movements remain unclear. KTN is a highly conserved cytoplasmic domain present ubiquitously in a variety of prokaryotic and eukaryotic K+ channels and transporters. Here we report crystal structures for two representative KTN domains that reveal a dimeric hinged assembly. Alternative ligands NAD+ and NADH block or vacate, respectively, the hinge region affecting the dimer's conformational flexibility. Conserved, surface-exposed hydrophobic patches that become coplanar upon hinge closure provide an assembly interface for KTN tetramerization. Mutational analysis using the KefC system demonstrates that this domain directly interacts with its respective transmembrane constituent, coupling ligand-mediated KTN conformational changes to the permease's activity.

Amino Acid Sequence↗