Pyrene Excimer Fluorescence of Yeast Alcohol Dehydrogenase: A Sensitive Probe to Investigate Ligand Binding and Unfolding Pathway of the Enzyme

Santra, Manas Kumar; Dasgupta, Debjani; Panda, Dulal

doi:10.1562/2005-09-26-ra-698

Cited by 14 publications

(5 citation statements)

References 35 publications

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“…Typically, pyrene is attached covalently to a naturally occurring or substituted Cys residue(s) to probe a specified site(s). Pyrene excimer fluorescence emission has been exploited to obtain information about the conformation and molecular organization of several proteins, − including apolipoproteins. − ,, In all these studies, a wide range of excimer emission intensities (relative to the intensity of band I of the monomeric ensemble) was noted and generally attributed to a distance of ∼10 Å between two probes. In this study, we investigated (i) the correlation between the distance between two pyrene-bearing locations and the extent of excimer fluorescence, (ii) the contribution of probe flexibility to excimer emission, and (iii) the organization of the linker segment of apoE in its tetrameric organization.…”

Section: Discussionmentioning

confidence: 99%

The Extent of Pyrene Excimer Fluorescence Emission Is a Reflector of Distance and Flexibility: Analysis of the Segment Linking the LDL Receptor-Binding and Tetramerization Domains of Apolipoprotein E3

et al. 2012

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Pyrene is a spatially sensitive probe that displays an ensemble of monomeric fluorescence emission peaks (375 – 405 nm), and an additional band (called excimer) at ~460 nm when two fluorophores are spatially proximal. We examined if there is a correlation between distance between two pyrenes on an α-helical structure and excimer/monomer (e/m) ratio. Using structure-guided design, pyrene maleimide was attached to pairs of Cys located in increments of ~ 5 Å apart on helix 2 of the N-terminal domain of apolipoprotein E3 (apoE3). Fluorescence spectral analysis revealed an intense excimer band when the probes were ~ 5 Å from each other with an e/m ratio of ~3.0, which decreased to ~1.0 at 20 Å. An inverse correlation between e/m ratio and the distance between pyrenes was observed, with the probe and helix flexibility also contributing to the extent of excimer formation. We verified this approach by estimating the distance between T57C and C112 (located on helices 2 and 3, respectively) to be 5.2 Å (4.9 Å from NMR and 5.7 Å from X-ray structure). Excimer formation was also noted to a significant extent with probes located in the linker segment suggesting spatial proximity (10 Å to 15 Å) from corresponding sites on neighboring molecules in the tetrameric configuration of apoE. We infer that oligomerization via the C-terminal domain juxtaposes the linker segments from neighboring apoE molecules. This study offers new insights into the conformation of tetrameric apoE and presents the use of pyrene as a powerful probe to study protein spatial organization.

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Section: Discussionmentioning

confidence: 99%

The Extent of Pyrene Excimer Fluorescence Emission Is a Reflector of Distance and Flexibility: Analysis of the Segment Linking the LDL Receptor-Binding and Tetramerization Domains of Apolipoprotein E3

et al. 2012

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“…Such theoretical evaluations are supported by rich experimental observations, where noticeable residual structure is seen in unfolded proteins even under the most severe denaturing conditions, such as high concentrations of strong denaturants. Among the illustrative examples of well‐characterized unfolded globular proteins with considerable residual structure are staphylococcal nuclease, the α‐subunit of tryptophan synthetase, fragment of the protein 434, human fibroblast growth factor 1, the SH3 domain, barstar, barnase, the WW‐domain, BPTI, chymotrypsin inhibitor 2, human carbonic anhydrase II apomyoglobin, lysozyme, photoactive yellow protein, the Escherichia coli outer membrane protein X, the N‐terminal domain of enzyme I from Streptomyces coelicolor , bovine and human α‐lactalbumins, protein eglin C, intestinal fatty acid binding protein, yeast alcohol dehydrogenase, HIV‐1 protease, “Trp‐cage” miniprotein TC5b, Bacillus licheniformis β‐lactamase, hyperthermophilic ribosomal protein S16, thermophilic ribonucleases H, and ubiquitin among many other examples.…”

Section: Diversity Of Conformational Ensembles Involved In Protein Fomentioning

confidence: 99%

Under‐folded proteins: Conformational ensembles and their roles in protein folding, function, and pathogenesis

Uversky

2013

Biopolymers

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For decades, protein function was intimately linked to the presence of a unique, aperiodic crystal-like structure in a functional protein. The two only places for conformational ensembles of under-folded (or partially folded) protein forms in this picture were either the end points of the protein denaturation processes or transiently populated folding intermediates. Recent years witnessed dramatic change in this perception and conformational ensembles, which the under-folded proteins are, have moved from the shadow. Accumulated to date data suggest that a protein can exist in at least three global forms-functional and folded, functional and intrinsically disordered (nonfolded), and nonfunctional and misfolded/aggregated. Under-folded protein states are crucial for each of these forms, serving as important folding intermediates of ordered proteins, or as functional states of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), or as pathology triggers of misfolded proteins. Based on these observations, conformational ensembles of under-folded proteins can be classified as transient (folding and misfolding intermediates) and permanent (IDPs and stable misfolded proteins). Permanently under-folded proteins can further be split into intentionally designed (IDPs and IDPRs) and unintentionally designed (misfolded proteins). Although intrinsic flexibility, dynamics, and pliability are crucial for all under-folded proteins, the different categories of under-foldedness are differently encoded in protein amino acid sequences.

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“…Based on 19 F NMR analysis it has been concluded that a residual structure in unfolded intestinal fatty acid-binding protein with incorporated fluorinated aromatic amino acids consists of amino acids that are neighbors in the native state (Ropson et al, 2006). In yeast alcohol dehydrogenase (YADH) with the cysteine residues covalently modified by N-(1-pyrenyl) maleimide (PM) residual structure was detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM-YADH (Santra et al, 2006). By a combination of circular dichroism (CD) and small-angle x-ray scattering (SAXS), some residual structure was found in the unfolded state of the HIV-1 protease (Kogo et al, 2009).…”

Section: Residual Structure In Proteins Unfolded By Strong Denaturantsmentioning

confidence: 99%

Fundamentals of Protein Folding

Uversky¹

2014

Protein Aggregation in Bacteria

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Pyrene Excimer Fluorescence of Yeast Alcohol Dehydrogenase: A Sensitive Probe to Investigate Ligand Binding and Unfolding Pathway of the Enzyme

Cited by 14 publications

References 35 publications

The Extent of Pyrene Excimer Fluorescence Emission Is a Reflector of Distance and Flexibility: Analysis of the Segment Linking the LDL Receptor-Binding and Tetramerization Domains of Apolipoprotein E3

The Extent of Pyrene Excimer Fluorescence Emission Is a Reflector of Distance and Flexibility: Analysis of the Segment Linking the LDL Receptor-Binding and Tetramerization Domains of Apolipoprotein E3

Under‐folded proteins: Conformational ensembles and their roles in protein folding, function, and pathogenesis

Fundamentals of Protein Folding

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