Solute perturbation of protein fluorescence. Quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion

Lehrer, Sherwin S.

doi:10.1021/bi00793a015

Cited by 1,771 publications

(1,146 citation statements)

References 42 publications

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“…These results demonstrate that the tryptophan accessibilities remain largely unaltered in the "molten globule-like" state of lysozyme in 25% HFA. In 6 M GdmCI, complete exposure of the tryptophan residues results in a dramatic enhancement of quenching efficiency in complete agreement with earlier results (Lehrer, 1971). Although disappearance of the near UV CD bands of lysozyme in 25% HFA shows disruption of specific native-like tertiary interactions, fluorescence quenching data strongly suggest that aromatic side chains of the tryptophan residues remain largely buried.…”

Section: Iodide Quenching Of Tryptophan Fluorescencesupporting

confidence: 91%

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Bhattacharjya

Balaram

1997

Protein Science

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A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced a f f i n i t y towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 'H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (250%). This transition is characterized by a dramatic loss of A N S binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. HID exchange experiments yield higher protection factors for many of the backbone amide protons from the four a-helices along with the C-terminal 3'0 helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel @-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for a-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

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Section: Iodide Quenching Of Tryptophan Fluorescencesupporting

confidence: 91%

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Bhattacharjya

Balaram

1997

Protein Science

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“…However, Fu equation was used for comparison in normalizing the denaturation curves of these mutants. Moreover, the changes in the fluorescence intensity only detect two states, including dissociation to monomers and unfolding of the monomer (Tominaga, 1994 and Ksv and [Q] are the same as above (Lehrer, 1971). The results in Figure 4B show that thef, values of W15, W320, and W64 in guanidine were approximately 1 , implying that these Trp residues were accessible to quencher.…”

Section: Iodide Quenchingmentioning

confidence: 90%

Enzymatic and fluorescence studies of four single‐tryptophan mutants of rat testis fructose 6‐phosphate,2‐kinase:fructose 2,6‐bisphosphatase

Watanabe

Jameson

Uyeda³

1996

Protein Science

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In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,Z-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1 . 5~ that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.

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“…The solvent exposure and accessibility of the tryptophan and the tyrosine on peptide fragment CB2 271-326 to the collisional quencher iodide was evaluated by Stern-Volmer analysis. Iodide is an ionic quencher and therefore will act only on fluorophores situated on the outer surface of proteins, and has been used for the quenching of proteins or peptides [9,32,34,35]. Stern-Volmer quenching for the tryptophan and the tyrosine on CB2 271-326 in iodide concentrations ranging from 0 to 200 mM was examined.…”

Section: Stern-volmer Quenchingmentioning

confidence: 99%

“…When excited at 275 nm (where both tyrosine and tryptophan residues absorb), the maximum emission wavelength was at 322 nm, which is an intermediate of the tryptophan (330 nm) and tyrosine (303 nm) residues. The steady-state fluorescence quenching data were analyzed using the Stern-Volmer equation [34]: …”

mentioning

confidence: 99%

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

Zhang

Xie

2008

Protein Expression and Purification

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Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2 , a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidineselected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2 , was subsequently purified by reverse-phase HPLC and confirmed by SDS-PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs. KeywordsCannabinoid receptor 2; transmembrane domain; inclusion body; affinity chromatography; HPLC; circular dichroism; fluorescence Since the discovery of the CB2 receptor in 1993 [1], there has been a growing interest to clarify the importance of this GPCR membrane protein in human physiology, and to investigate it as a possible target for current and future drug development. The CB2 receptor seems to have a significant role in mediating the immunological functions of cannabinoid receptor ligands. Thus, the CB2 receptor is a promising target for drug development that aims at management of neurological pain, inflammation, osteoporosis, and growth of malignant gliomas and tumors * Corresponding author. Fax: +1 412 383 5276. E-mail address: xix15@pitt.edu (X-Q. Xie). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [3][4][5][6][7]. In general, the Escherichia coli expression system is the most attractive strategy due to its advantages of simplicity of use, low cost, easy scale-up, and homogeneous protein production. Though few attempts were promising to express full length CB2 receptor in ...

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Solute perturbation of protein fluorescence. Quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion

Cited by 1,771 publications

References 42 publications

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Enzymatic and fluorescence studies of four single‐tryptophan mutants of rat testis fructose 6‐phosphate,2‐kinase:fructose 2,6‐bisphosphatase

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

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