Using an Amino Acid Fluorescence Resonance Energy Transfer Pair To Probe Protein Unfolding: Application to the Villin Headpiece Subdomain and the LysM Domain

Glasscock, Julie M.; Zhu, Yili; Chowdhury, P. K.; Jia, Tao; Gai, Feng

doi:10.1021/bi8012406

Cited by 61 publications

(75 citation statements)

References 52 publications

(85 reference statements)

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“…The method will be easiest to interpret quantitatively in terms of helix formation when the F CN group is exposed to solvent in both states since the fluorescence quantum yield is influenced by hydrogen bonding to the cyano group as well as by quenching from other protein side-chains. If the F CN group were buried in one conformation, but not in the other, then the observed intensity change would be a convolution of the effects due to changes in side-chain quencher interactions and the effects of changes in solvation (18). However, note that both quenching and side-chain burial reduce F CN fluorescence.…”

Section: Resultsmentioning

confidence: 99%

Modulation of p-Cyanophenylalanine Fluorescence by Amino Acid Side Chains and Rational Design of Fluorescence Probes of α-Helix Formation

et al. 2010

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p-Cyanophenylalanine is an extremely useful fluorescence probe of protein structure which can be recombinantly and chemically incorporated into proteins. The probe has been used to study protein folding, protein-membrane interactions, protein-peptide interactions and amyloid formation, however the factors that control its fluorescence are not fully understood. Hydrogen bonding to the cyano group is known to play a major role in modulating the fluorescence quantum yield, but the role of potential side-chain quenchers has not yet been elucidated. A systematic study on the effects of different side-chains on p-cyanophenylalanine fluorescence is reported. Tyr is found to have the largest effect followed by deprotonated His, Met, Cys, protonated His, Asn, Arg, and protonated Lys. Deprotonated amino groups are much more effective fluorescence quenchers than protonated amino groups. Free neutral imidazole and hydroxide ion are also effective quenchers of p-cyanophenylalanine fluorescence with Stern-Volmer constants of 39.8 M−1 and 22.1 M−1, respectively. The quenching of p-cyanophenylalanine fluorescence by specific side-chains is exploited to develop specific, high sensitivity, fluorescence probes of helix formation. The approach is demonstrated with Ala based peptides that contain a p-cyanophenylalanine-His or a p-cyanophenylalanine-Tyr pair located at positions i and i+4. The p-cyanophenylalanine-His pair is most useful when the His side-chain is deprotonated and is, thus, complimentary to Trp-His pair which is most sensitive when the His side-chain is protonated.

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Section: Resultsmentioning

confidence: 99%

Modulation of p-Cyanophenylalanine Fluorescence by Amino Acid Side Chains and Rational Design of Fluorescence Probes of α-Helix Formation

et al. 2010

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“…HP35 folds fast (∼1 μs) compared to large proteins, allowing tractable folding/unfolding simulations. Consequently, it has served as a model system in a number of computational folding studies (3)(4)(5), as well as a variety of experimental studies (6)(7)(8). NMR and X-ray diffraction studies have indicated that wild-type HP35 folds into three alpha helices ( Fig.…”

mentioning

confidence: 99%

Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopy

Chung

Thielges

Fayer

2011

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

108

166

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A series of two-dimensional infrared vibrational echo experiments performed on nitrile-labeled villin headpiece [HP35-ðCNÞ 2 ] is described. HP35 is a small peptide composed of three alpha helices in the folded configuration. The dynamics of the folded HP35-ðCNÞ 2 are compared to that of the guanidine-induced unfolded peptide, as well as the nitrile-functionalized phenylalanine (PheCN), which is used to differentiate the peptide dynamic contributions to the observables from those of the water solvent. Because the viscosity of solvent has a significant effect on fast dynamics, the viscosity of the solvent is held constant by adding glycerol. For the folded peptide, the addition of glycerol to the water solvent causes observable slowing of the peptide's dynamics. Holding the viscosity constant as GuHCl is added, the dynamics of unfolded peptide are much faster than those of the folded peptide, and they are very similar to that of PheCN. These observations indicate that the local environment of the nitrile in the unfolded peptide resembles that of PheCN, and the dynamics probed by the CN are dominated by the fluctuations of the solvent molecules, in contrast to the observations on the folded peptide.nitrile IR probe | peptide dynamics T he structure of proteins, both folded and unfolded, are inherently dynamic in nature. A protein is constantly undergoing interconversions among a range of structures separated by relatively low energy barriers. Fast conformational sampling can give rise to protein structural evolution on slower time scales. Many experimental and theoretical studies have focused on the native structure due to the relevance of the native state protein to its biological functions, but also because of difficulties associated with characterizing proteins under unfolding conditions, where they can exist in heterogeneous distributions of many conformations (1, 2). Properties of the unfolded state are also significant because they play important roles in folding and stability, transport across membranes, and proteolysis and protein turnover (1). Unfolded proteins have dynamics that are different from the native protein, and the differences in dynamics can shed light on the relationship between the native and the unfolded protein.An attractive target for studying differences between the folded and unfolded structures is the villin headpiece 35 (HP35), a peptide chain composed of 35 amino acids found in a chicken villin as a small subdomain. HP35 folds fast (∼1 μs) compared to large proteins, allowing tractable folding/unfolding simulations. Consequently, it has served as a model system in a number of computational folding studies (3-5), as well as a variety of experimental studies (6-8). NMR and X-ray diffraction studies have indicated that wild-type HP35 folds into three alpha helices (Fig. 1), which encase the hydrophobic core composed of three phenylalanines (9, 10).In this paper, the results of a series of ultrafast 2D IR vibrational echo experiments on nitrile-incorporated HP35 are presented. Two CN-fun...

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“…However, Kayser et al found it to be a particularly useful tool for studying structure-function relationships in monoclonal antibodies, which were found to have an unusually high Trp content [199]. Another use of Trp as a natural fluorophore for FRET is to probe protein folding/unfolding, an important yet poorly understood biological process [200][201][202]. For example, Jha et al used FRET to study the unfolding of a small protein, monellin [202].…”

Section: Natural Fluorophoresmentioning

confidence: 99%

“…The photophysics of this useful nonnatural amino acid has recently been characterized [211,215]. The Trp-PheCN FRET pair has been used to determine detailed protein folding and unfolding in two small proteins, the villin headpiece subdomain (HP35) and the lysin motif (LysM) domain [200]. Two other nonnatural amino acids, 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) ( Figure 6.13), were recently found to be FRET acceptors to PheCN [210].…”

Section: Nonnatural Amino Acidsmentioning

confidence: 99%

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

Sapsford

Wildt

Mariani

et al. 2013

FRET – Förster Resonance Energy Transfer

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The intrinsic sensitivity (r 6 dependence) of F€ orster (or fluorescence) resonance energy transfer (FRET) to nanoscale changes in the donor/acceptor separation distance has made FRET an invaluable biophysical tool in a variety of applications, ranging from studying the structure and conformation of proteins and nucleic acids to examining biomolecular interactions, including its use in in vitro and in vivo bioassays [1][2][3][4][5][6][7]. While the myriad of FRET configurations and techniques currently in use are covered throughout this book, here we focus primarily on the materials utilized as donor or acceptor probes in FRET rather than the process itself [3,5,8,9]. Our 2006 review paper on this topic serves as the foundation for this updated chapter [3]. The materials were divided into three main categories: organic materials that include "traditional" dye fluorophores, dark quenchers, polymers, and carbon nanomaterials (NMs); inorganic materials such as metal chelates, metal, and semiconductor nanocrystals; and fluorophores of biological origin such as fluorescent proteins (FPs), amino acids, and fluorescence generated from enzymatic bioluminescence (BL) and chemiluminescence (CL). These materials may function as FRET donors and/or acceptors, depending upon experimental design. Many of the new materials developed and/or new donor-acceptor probe combinations used address some of the inherent complications of more traditional FRET materials, including photobleaching, spectral cross talk, and direct excitation of the acceptor species, and examples of these will be highlighted and discussed throughout the chapter. Since the vast majority of FRET applications are biological in nature, they routinely involve some type of biomolecule labeling strategy, which ultimately plays a significant and fundamental role in the success and interpretation of the resulting FRET. Therefore, we begin the chapter with a brief discussion of the bioconjugation techniques commonly utilized for FRET and points to consider, followed by sections highlighting the current and emerging materials that are used, or have the potential to be used, in FRET applications.

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Using an Amino Acid Fluorescence Resonance Energy Transfer Pair To Probe Protein Unfolding: Application to the Villin Headpiece Subdomain and the LysM Domain

Cited by 61 publications

References 52 publications

Modulation of p-Cyanophenylalanine Fluorescence by Amino Acid Side Chains and Rational Design of Fluorescence Probes of α-Helix Formation

Modulation of p-Cyanophenylalanine Fluorescence by Amino Acid Side Chains and Rational Design of Fluorescence Probes of α-Helix Formation

Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopy

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

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