Simple and Efficient Strategy for Site-Specific Dual Labeling of Proteins for Single-Molecule Fluorescence Resonance Energy Transfer Analysis

Kim, Jihyo; Seo, Myung Won; Lee, Sangsik; Cho, Kyukwang; Yang, Aerin; Woo, Kyunghwa; Kim, Kwang Ho; Park, Hee-Sung

doi:10.1021/ac303089v

Cited by 47 publications

(35 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We attempted to directly assess RecBCD subunit movement during its reaction on DNA by using Förster resonance energy transfer, but we were unable to synthesize sufficient RecBCD to allow incorporation of fluorescent moieties into enzyme containing two non-natural amino acids [47] (unpublished data). We wished to test a mutant RecBCD lacking the nuclease domain, but this enzyme proved to be too unstable for these biochemical experiments (unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Taylor

Amundsen

Guttman

et al. 2014

Journal of Molecular Biology

View full text Add to dashboard Cite

Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5′ GCTGGTGG 3′) during DNA unwinding, RecBCD’s unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD’s DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3′-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular “signal transduction” from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi’s stimulation of recombination.

show abstract

Section: Discussionmentioning

confidence: 99%

Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Taylor

Amundsen

Guttman

et al. 2014

Journal of Molecular Biology

View full text Add to dashboard Cite

show abstract

“…Structural changes in different regions of a protein also may be combined with dynamic interfacial, as well as functional, measurements, allowing direct conclusions to be drawn with respect to ways in which protein conformation, dynamics, and activity are connected. Although site-specific labeling methods have been used with FRET to monitor conformational changes in single protein molecules in bulk solution (17)(18)(19) or covalently tethered to surfaces (20)(21)(22), such methods have not been applied to the analysis of proteins undergoing dynamic adsorption, desorption, and diffusion while interacting directly with material interfaces. SM-FRET tracking, with temporal resolution of 100 ms, has previously been used to monitor dynamic changes in conformation of mobile DNA at the solution-surface interface (23,24), using high-throughput tracking algorithms that permit the observation of 10 4 -10 6 molecules.…”

Section: Significancementioning

confidence: 99%

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

McLoughlin

Kastantin

Schwartz

et al. 2013

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

View full text Add to dashboard Cite

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.protein adhesion | single-molecule fluorescence | total internal reflection fluorescence microscopy U nderstanding the effect of near-surface environments on protein conformation is critical in many bioengineering and biomedical applications, including biosensing, cell culture, tissue engineering, biocatalysis, and pharmaceutical formulation. Importantly, surface interactions that perturb protein structure can inactivate proteins, as has widely been observed in the case of surface-immobilized enzymes (1-6). Such interactions, by inducing unfolding and subsequent accumulation of freely absorbing proteins on biomaterial surfaces, may trigger unfavorable cellular responses (7-10). However, experimental methods to elucidate both protein structure and interfacial dynamics (e.g., adsorption, diffusion, desorption), particularly in heterogeneous near-surface environments, are virtually nonexistent.Conventional methods to determine surface effects on protein structure are largely ensemble-averaging techniques that provide limited mechanistic insight. Such limitations can lead to misinterpretations of apparent interfacial phenomena, which are used to explain the biocompatibility of biomaterials. For example, conventional biophysical methods often find that the average surface protein conformation relaxes from ...

show abstract

“…A study by Park and colleagues used amber/opal (UAG/UGA) suppression to incorporate N 6 -propargyloxycarbonyl-lysine (Pok) and p -acetylphenylalanine (AcF) into CaM. [38] The dual-functionalized CaM was labeled in one pot using Cy3-hydrazide (Acf) and Cy5-azide (Pok), and the resulting construct was analyzed by ensemble and single molecule FRET (smFRET) in the presence or absence of Ca 2+ and M13 binding peptide. However, the efficiency of dual suppression techniques like these is limited by truncation at the UAG codon due to competition with release factors or incorporation of natural amino acids in response to the suppressed codon.…”

Section: Dual Uaa Labelingmentioning

confidence: 99%

Multiply labeling proteins for studies of folding and stability

Haney

Wissner

Petersson

2015

Current Opinion in Chemical Biology

View full text Add to dashboard Cite

Fluorescence spectroscopy is a powerful method for monitoring protein folding in real-time with high resolution and sensitivity, but requires the site-specific introduction of labels into the protein. The ability to genetically incorporate unnatural amino acids allows for the efficient synthesis of fluorescently-labeled proteins with minimally perturbing fluorophores. Here, we describe recent uses of labeled proteins in dynamic structure determination experiments and advances in unnatural amino acid incorporation for dual site-specific fluorescent labeling. The advent of increasingly sophisticated bioorthogonal chemistry reactions and the diversity of unnatural amino acids available for incorporation will greatly enable protein folding and stability studies.

show abstract

Simple and Efficient Strategy for Site-Specific Dual Labeling of Proteins for Single-Molecule Fluorescence Resonance Energy Transfer Analysis

Cited by 47 publications

References 23 publications

Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

Multiply labeling proteins for studies of folding and stability

Contact Info

Product

Resources

About