Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science. 282:2220-2226), Gly(22) is buried within the constriction that closes the pore. Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.
We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.
MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock. We have previously shown that substitution of a residue that resides within the channel pore constriction, MscL's Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution (). Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp. Binding MTS reagents that are positively charged ([2-(trimethylammonium)ethyl] methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied. In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold. These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study. In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state. These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.
Ca 2؉ is released from the vacuole into the yeast cytoplasm on an osmotic upshock, but how this upshock is perceived was unknown. We found the vacuolar channel, Yvc1p, to be mechanosensitive, showing that the Ca 2؉ conduit is also the sensing molecule. Although fragile, the yeast vacuole allows limited direct mechanical examination. Pressures at tens of millimeters of Hg (1 mmHg ؍ 133 Pa) activate the 400-pS Yvc1p conductance in whole-vacuole recording mode as well as in the excised cytoplasmic-side-out mode. Raising the bath osmolarity activates this channel and causes vacuolar shrinkage and deformation. It appears that, on upshock, a transient osmotic force activates Yvc1p to release Ca 2؉ from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca 2؉ concentration and is apparently independent of its known Ca 2؉ activation, which we now propose to be an amplification mechanism (Ca 2؉ -induced Ca 2؉ release). Yvc1p is a member of the transient receptor potential-family channels, several of which have been associated with mechanosensation in animals. The possible use of Yvc1p as a molecular model to study mechanosensation in general is discussed.
The technology now exists to construct physical models of proteins based on atomic coordinates of solved structures. We review here our recent experiences in using physical models to teach concepts of protein structure and function at both the high school and the undergraduate levels. At the high school level, physical models are used in a professional development program targeted to biology and chemistry teachers. This program has recently been expanded to include two student enrichment programs in which high school students participate in physical protein modeling activities. At the undergraduate level, we are currently exploring the usefulness of physical models in communicating concepts of protein structure and function that have been traditionally difficult to teach. We discuss our recent experience with two such examples: the close-packed nature of an enzyme active site and the pH-induced conformational change of the influenza hemagglutinin protein during virus infection.A common goal of biochemistry educators is to provide students with a deep understanding of fundamental concepts underlying protein structure and function. This is most commonly done by exposing students to stunning two-dimensional color graphics of proteins in textbooks and frequently augmenting these static figures with interactive images that can be rotated in three-dimensional space in a computer environment. Although this approach is successful for those students who are able to infer three-dimensional information from these inherently twodimensional representations, many other students fail to make this inference. For them, the molecular world of proteins remains an abstraction for which they have little interest. We have found that physical models of proteins ( Fig. 1) are amazingly effective tools that initially capture the interest of this larger group of students and motivate them to learn more about this invisible, molecular world. These physical models are synergistic with computer visualization tools, allowing students to generalize their initial understanding of a specific protein to other structures that are explored in a computer environment. We review here our recent experience with the use of physical models to make this molecular world "real" for students at both the high school and the undergraduate levels. A THEORETICAL BASIS FOR THE VALUE OF PHYSICAL MODELS IN TEACHING ABSTRACT CONCEPTS IN SCIENCEThe value of physical models of small molecules in organic chemistry courses is well known to biochemistry educators. However, these small molecule kits are not practical for modeling the higher order molecular structures of proteins. Experienced researchers have learned to infer three-dimensional information from two-dimensional images of proteins or to manipulate interactive, computergenerated images of proteins. Unfortunately, our current educational practice treats inexpert students as though they were expert researchers. Students are introduced to proteins through two-dimensional drawings or interactive computer visualiz...
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