Single-Molecule Surface Enhanced Resonance Raman Spectroscopy of the Enhanced Green Fluorescent Protein

Habuchi, Satoshi; Cotlet, Mircea; Gronheid, Roel; Dirix, Gunter; Michiels, Jan; Vanderleyden, Jos; Schryver, and Frans C. De; Hofkens, Johan

doi:10.1021/ja0353311

Cited by 146 publications

(132 citation statements)

References 29 publications

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“…The nature of these dark states is still not clear, although changes in the protonation state of the chromophore have been suggested [46,97]. In a report using surface-enhanced resonance Raman measurements, the signatures of the protonated and deprotonated chromophore were found to change on time scales similar to that of fluorescence blinking [101]. Nevertheless, the attribution of blinking to changes in the protonation state has been questioned by the finding that the blinking rates on the millisecond to second time scale were insensitive to pH over a large range [98].…”

Section: Single Vfp Intensity Trajectoriesmentioning

confidence: 99%

Single-molecule spectroscopy of fluorescent proteins

Blum

Subramaniam

2008

Anal Bioanal Chem

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The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the details of the photophysics of these reporters is essential for accurate interpretation of the biological and biochemical processes illuminated by fluorescent proteins. Some aspects of the complex photophysics of fluorescent proteins can only be observed and understood at the singlemolecule level, which removes averaging inherent to ensemble studies. In this paper we review how singlemolecule emission detection has helped understanding of the complex photophysics of fluorescent proteins.

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Section: Single Vfp Intensity Trajectoriesmentioning

confidence: 99%

Single-molecule spectroscopy of fluorescent proteins

Blum

Subramaniam

2008

Anal Bioanal Chem

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“…The protein GFP was studied using SERRS at the single molecule level [30], and in this case it was observed that the spectrum changes with time. This spectral change was also observed with SERS studies on Cytochrome C extracted from yeast [31].…”

Section: Sers Of Large Biological Moleculesmentioning

confidence: 99%

Surface enhanced Raman scattering for narcotic detection and applications to chemical biology

Ryder

2005

Current Opinion in Chemical Biology

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Raman spectroscopy is rapidly finding favour for applications in the life science due to the ease with which it can be used to extract significant data from tissue and cells. However, the Raman effect is an inherently weak effect, which hinders the analysis of low concentration analytes. The Raman sensitivity can be improved via the Surface Enhanced Raman Scattering (SERS) effect. In SERS, Raman spectra are dramatically increased when a molecule is adsorbed onto nanoroughened noble metal surfaces such as silver and gold. The degree of enhancement enables single molecule detection, which offers the potential for the unambiguous identification of analytes at concentrations that are useful in both a forensic and a chemical biology context. Here we discuss some of the practical applications of SERS to both low-level narcotic detection, and how this can be applied to chemical biology.

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“…The EGFP-rhodanese chimera was generated by digesting the PCR product with HindIII and inserting it into the pBAD/HisA(EGFP) vector that was previously digested by the same enzyme, treated with calf intestinal alkaline phosphatase, and purified. The pBAD/HisA(EGFP) vector was previously constructed by removing the gene for EGFP(Q185H) from pEGFP (Clontech, Saint-Germain-en-Laye, France) into the PstI/EcoRI sites of pBAD/HisA (Invitrogen) as described (25), thereby adding to the protein an N-terminal tail that contains a His 6 -tag. A stop codon between EGFP and rhodanese was removed by using site-directed mutagenesis performed with a QuikChange kit (Stratagene, La Jolla, CA) and the oligonucleotides 5Ј-CGGCATGGACGAGCTGTACA AG*A A A-GCGGCCGCGACTCTAGA AT TCG-3Ј (forward) and 5Ј-CGAATTCTAGAGTCGCGGCCGCTTT*CTTGTACAG-CTCGTCCATGCCG-3Ј (reverse).…”

Section: Methodsmentioning

confidence: 99%

Concerted ATP-induced allosteric transitions in GroEL facilitate release of protein substrate domains in an all-or-none manner

Kipnis¹,

Papo²,

Haran

et al. 2007

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

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The double-ring chaperonin GroEL mediates protein folding, in conjunction with its helper protein GroES, by undergoing ATPinduced conformational changes that are concerted within each heptameric ring. Here we have examined whether the concerted nature of these transitions is responsible for protein substrate release in an all-or-none manner. Two chimeric substrates were designed, each with two different reporter activities that were recovered after denaturation in GroES-dependent and independent fashions, respectively. The refolding of the chimeras was monitored in the presence of GroEL variants that undergo ATPinduced intraring conformational changes that are either sequential (F44W/D155A) or concerted (F44W). Our results show that release of a protein substrate from GroEL in a domain-by-domain fashion is favored when the intraring allosteric transitions of GroEL are sequential and not concerted.allostery ͉ chaperonins ͉ cooperativity ͉ protein folding T he Escherichia coli chaperonin GroEL is a molecular machine that assists protein folding by undergoing allosteric transitions between protein substrate binding and release states (for reviews, see refs. 1-3). It is made up of two homoheptameric rings, stacked back-to-back, with a cavity at each end (4) in which protein folding can take place in a confining and protective environment. GroEL functions in conjunction with a heptameric ring-shaped cochaperonin, GroES (5), that caps the cavity of the so-called cis ring (6), thereby triggering dissociation of bound protein substrates into the cavity. The allosteric transitions of GroEL are induced by ATP binding that occurs with positive cooperativity within rings and negative cooperativity between rings (7, 8). Experimental data (refs. 9 and 10 and G. Curien, J. Grason, and G. Lorimer, personal communication) and simulations (11) have shown that the intraring allosteric transitions of GroEL are concerted, in accordance with the Monod-WymanChangeux model of cooperativity (12), as proposed in the nested model (7). In contrast, the intraring allosteric transitions of the eukaryotic heterooligomeric chaperonin CCT were recently shown to occur in a sequential fashion (13) in accordance with the Koshland-Némethy-Filmer model of cooperativity (14). The implications of these different allosteric mechanisms for the folding function of chaperonins have not yet been explored.The work described here was undertaken to establish whether the concerted intraring ATP-induced allosteric transitions of GroEL facilitate simultaneous release of different parts of a bound protein substrate, as previously speculated (13). By contrast, it was suggested (13) that ATP-induced sequential conformational changes in CCT may facilitate sequential release and, as a result, domain-by-domain substrate folding. A powerful tool available for investigating this question is a wild-type variant of GroEL that contains a fluorescence reporter introduced by the F44W mutation and has the D155A substitution that converts its ATP-induced intraring allosteric t...

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Single-Molecule Surface Enhanced Resonance Raman Spectroscopy of the Enhanced Green Fluorescent Protein

Cited by 146 publications

References 29 publications

Single-molecule spectroscopy of fluorescent proteins

Single-molecule spectroscopy of fluorescent proteins

Surface enhanced Raman scattering for narcotic detection and applications to chemical biology

Concerted ATP-induced allosteric transitions in GroEL facilitate release of protein substrate domains in an all-or-none manner

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