Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

Krueger, Andrew T.; Imperiali, Barbara

doi:10.1002/cbic.201300079

Cited by 92 publications

(57 citation statements)

References 101 publications

(52 reference statements)

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“…One approach using a much smaller fluorescent tag involves fluorescent labeling of SNAREs with the biarsenical dye FlAsH using the tetracysteine system (1,64). Recent progress in the incorporation of fluorescent unnatural amino acids in proteins expressed in mammalian cells (29) suggests that this method may be another possible tool to provide more detailed insight into the conformational changes of the SNARE complex that lead to fusion pore formation. These may also be able to provide direct evidence regarding the controversy if a stable, …”

Section: The Mechanism By Which the Force Transfer Leads To Fusion Pomentioning

confidence: 99%

How Could SNARE Proteins Open a Fusion Pore?

Fang

Lindau

2014

Physiology

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The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion.In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains.Qinghua Fang 1,2 and Manfred Lindau (49) has three components. The v-SNARE synaptobrevin 2 (syb2 also called VAMP2) is a 116-amino acid protein anchored in the vesicle membrane by a single transmembrane (TM) domain. Syntaxin (stx) is correspondingly anchored in the plasma membrane via a single TM helix. The third component, SNAP-25, has lipid anchors in the plasma membrane. SNAP-25 and stx are called t-SNAREs, being in the target membrane for fusion of secretory vesicles. The key importance of these proteins in the fusion mechanism has been demonstrated by the finding that proteolytic cleavage of the SNARE proteins by specific neurotoxins results in strong inhibition of transmitter release in neurons as well as in chromaffin cells (42,44). One example for the medical relevance of the SNARE complex is the BoTox treatment, which exerts its function through inhibition of transmitter release by specific cleavage of the SNARE protein SNAP-25. The cytosolic so-called SNARE domains of these three proteins form a coiled coil, which incorporates one helix each from syb2 and stx and two helices (named SN1 and SN2) from SNAP-25. The coiled coil structure of the SNARE domains has been solved by X-ray crystallography several years ago (57). Based on this structure, the configuration shown in FIGURE 1A was proposed for a trans configuration, in which a SNARE complex links the vesicle membrane and plasma membrane. The arrows indicate the cleavage sites for the neurotoxins mentioned above. More recently, a crystal structure of the SNARE complex, including the syb2 and stx TM helices, was solved (56), which shows helical extension from the SNARE domains through the linkers into the TM domains (FIGURE 1B). This structure is thought to resemble a post-fusion state in which all components of the SNARE complex are in a cis configuration, located in the same fused membrane.Synaptic vesicles contain ϳ70 copies of syb2 (59), but it is unknown and controversial how many of these copies are acting together in the formation of a fusion pore (43). Interestingly, neurosecretory vesicles in PC 12 cells recruit t-SNARE clusters containing a similar number of stx and SNAP-25 molecules (26). It has been shown that in reconstituted systems a single SNARE complex is sufficient to promote lipid vesicle fusion (50, 62). However, at least three SNARE complexes appear to be required for fast membrane fusion kinetics (8,17,41,50). In cultured hippocampal neurons, two copies of syb2 were found to be sufficient for fusion, but the experiments were performed with low time resolution (52). The Triggering MechanismTransmitter rel...

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Section: The Mechanism By Which the Force Transfer Leads To Fusion Pomentioning

confidence: 99%

How Could SNARE Proteins Open a Fusion Pore?

Fang

Lindau

2014

Physiology

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“…We observed high levels of scaffold expression and unnatural amino acid incorporation with no apparent azide photolysis based on high resolution ESI mass spectrometry (Fig. 1C), despite A50 and A199 being located on the protein interior [23] . The hexa-histidine tagged scaffold proteins were purified by Ni-affinity chromatography following an initial heat treatment, [20] and both fluorescence (Fig.…”

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confidence: 99%

A General Method for Artificial Metalloenzyme Formation through Strain‐Promoted Azide–Alkyne Cycloaddition

et al. 2013

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Strain-promoted azide-alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p-azido-L-phenylalanine (Az) residue and catalytically active bicyclononyne-substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor-based ArM formation allows the use of any desired metal complex to build unique inorganic-protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process since it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both Si-H and olefin insertion reactions involving these carbene precursors.

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“…Moreover, studies in mice indicated that the rFX386Cys and rFXwt molecules behaved similarly in terms of recovery. These features make the rFX386Cys variant a potential target for thiol-specific ligands for study in mice using established models such as intravital microscopy [39,40].…”

Section: Discussionmentioning

confidence: 99%