Excitatory signaling in bacterial probed by caged chemoeffectors

Khan, Shahid; Castellano, Fred; Spudich, Jhn L.; McCray, James A.; Goody, Roger S.; Reid, Gordon P.; Trentham, David R.

doi:10.1016/s0006-3495(93)81317-1

Cited by 86 publications

(118 citation statements)

References 77 publications

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“…The response time of E. coli cells to stimuli is generally found to be Ϸ50-100 ms (36,37). Although large changes in membrane architecture may not occur on this time scale, local movements could permit additional CheA and receptor proteins near the edge of an active receptor͞signaling complex to join into it and thus increase the net CheA activity.…”

Section: Discussionmentioning

confidence: 99%

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

Wolanin¹,

Baker

Francis³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Escherichia coli chemotaxis is mediated by membrane receptor͞ histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, lzTar C, that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from lzTar C. Thus, the organization of these receptor͞signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo.CheA ͉ histidine kinase ͉ serine receptor ͉ signal transduction

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Section: Discussionmentioning

confidence: 99%

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

Wolanin¹,

Baker

Francis³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The techniques for caging essentially any biomolecule or second messenger have now been developed, so that protons 60 , inorganic cations, gases 61 , small organic molecules and macromolecules 46 , 62 can be caged. But given the complexity of the syntheses of most caged compounds, commercial availability constrains their use for most laboratories.…”

Section: Caged Mrna and Dnamentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group (Fig. 1). Illumination results in a concentration jump of the biologically active molecule that can bind to its cellular receptor, switching on (or off) the targeted process. Virtually every kind of signaling molecule or second messenger, of every size| [mdash]|from protons to proteins| [mdash]|has been caged 1 .Why are caged compounds so useful? A single component of cellular chemistry can control the function of a cell, and such cellular regulation can be temporally or spatially defined, intracellular or extracellular, and amplitude-or frequency-modulated 2 . Photomanipulation of cellular chemistry using caged compounds provides a uniquely powerful means to interact with such cellular dynamics, as it can touch upon any one of the above dimensions. Thus, since light passes through cell membranes, uncaging can rapidly release a biomolecule in an intracellular compartment. This space is not readily accessible to many second messengers (for example, inositol-1,4,5-trisphosphate (IP 3 ), ATP, Ca 2+ , cAMP, cGMP) when they are applied to cells externally, as their charge makes them impermeable to the plasma membrane. Furthermore, uniform illumination results in release throughout the cytosol, or the release can be localized by focusing the uncaging beam on one part of a cell. Likewise, extracellular uncaging of neurotransmitters and hormones is tunable, allowing stimulation of many neurons simultaneously or of single synapses| [mdash]|by global or focused illumination, respectively. Light cannot only be directed, but also modulated in time andCorrespondence should be addressed to Graham C R Ellis-Davies (cagedca@hotmail.com). (Fig. 2) has been widely used to study many Ca 2+ -controlled processes. In particular, secretory processes in neuronal and non-neuronal cells have been extensively studied with caged calcium 22 . Rapid uncaging of glutamate using two-photon photolysis is particularly useful for the rational stimulation of visually identified synapses in complex tissue preparations such as acutely isolated brain slices from the hippocampus 23 (Fig. 2b). Finally, uncaging of mRNA in vivo in zebrafish is an excellen...

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“…For example, caged dioxygen (MacArthur et al, 1995), which displays a quantum yield of 0.04 at physiological pH, could serve to study the mechanisms of many dioxygenases. Caged protons (Khan et al, 1993) could also be used to trigger pH-dependent catalytic activity at low temperature.…”

Section: Discussionmentioning

confidence: 99%

“…In the latter case, the system must be brought after cryophotolysis to a temperature that permits diffusion of the uncaged high-af®nity compound from the solvent channels into the active site. This is expected to occur just above the glass-transition temperature (Smith & Kay, 1999) for small molecules such as dioxygen (MacArthur et al, 1995) or protons (Khan et al, 1993) and possibly also for larger molecules such as ATP. In the former case, the temperature increase may need to allow the released cage to diffuse out of the active site.…”

Section: Discussionmentioning

confidence: 99%

Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals

Ursby

Weik

Fioravanti

et al. 2002

Acta Crystallogr D Biol Cryst

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Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s À1 ) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in¯ash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100±150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryophotolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.

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Excitatory signaling in bacterial probed by caged chemoeffectors

Cited by 86 publications

References 77 publications

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals

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