Folding dynamics of single GCN-4 peptides by fluorescence resonant energy transfer confocal microscopy

Jia, Yiwei; Talaga, David; Lau, Wai Leung; Lu, Helen S. M.; DeGrado, William F.; Hochstrasser, Robin M.

doi:10.1016/s0301-0104(99)00127-5

Cited by 153 publications

(106 citation statements)

References 34 publications

Supporting

Mentioning

102

Contrasting

Unclassified

Order By: Relevance

“…These results, as well as the previous work on proteins unfolded by chemical denaturants (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), suggests that single-molecule FRET will become an increasingly powerful tool in investigations of structure distributions in protein folding and related problems.…”

Section: Discussionmentioning

confidence: 52%

“…This development has enabled several single-molecule FRET studies of protein folding, aimed at obtaining information on intramolecular distance distributions as molecules fold (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). As a ''control'' for determining the accuracy of distances obtained from single-molecule FRET results, Schuler et al (15) effectively repeated the Stryer and Haugland experiment at the single-molecule level on freely diffusing molecules with continuous wave laser excitation (15), using longer polyprolines of 6-40 residues because of the larger R 0 (5.4 nm) in their experiment.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline

Best

Merchant

Gopich

et al. 2007

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

208

297

View full text Add to dashboard Cite

Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Fö rster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to the flexibility of the polypeptide. Here, we investigate this issue further, using a combination of single-molecule fluorescence intensity and lifetime measurements, NMR, theory, and molecular dynamics simulations of polyproline-20 that include the dyes and their linkers to the polypeptide. NMR shows that in water Ϸ30% of the molecules contain internal cis prolines, whereas none are detectable in trifluoroethanol. Simulations suggest that the all-trans form of polyproline is relatively stiff, with persistence lengths of 9 -13 nm using different established force fields, and that the kinks arising from internal cis prolines are primarily responsible for the higher mean FRET efficiency in water. We show that the observed efficiency histograms and distributions of donor fluorescence lifetimes are explained by the presence of multiple species with efficiencies consistent with the simulations and populations determined by NMR. In calculating FRET efficiencies from the simulation, we find that the fluctuations of the chromophores, attached to long flexible linkers, also play an important role. A similar simulation approach suggests that the flexibility of the chromophore linkers is largely responsible for the previously unexplained high value of R 0 required to fit the data in the classic study of Stryer and Haugland.fluorescence ͉ molecular dynamics ͉ persistence length ͉ polypeptide ͉ proteins F örster resonance energy transfer (FRET) has been a widely used tool for determining distances within and between biological molecules, and was called a ''spectroscopic ruler'' by Stryer and Haugland (1, 2). The basis of the technique is that the rate of energy transfer, k ET , between two chromophores depends on their separation in space R according to k ET ϭ k D (R 0 /R) 6 , as shown by Förster (k D Ϫ1 is the fluorescence lifetime of the ''donor'' chromophore and R 0 the distance at 50% transfer efficiency). The prediction of an R Ϫ6 dependence of k ET was initially confirmed in the experiment by Stryer and Haugland (1), who used a series of L-proline oligomers (Fig. 1A) as spacers of known length (3) between naphthyl and dansyl chromophores. Based on the early theoretical estimate by Schimmel and Flory (4) of 22 nm for the persistence length of poly-L-proline in the type II conformation, the spacers of 1-12 prolines were considered as rigid rods, resulting in a good fit of the theory to the experimental transfer efficiencies. An unexplained result, however, was that the fit required an R 0 significantly longer than the value determined spectroscopically.Advances in detector technology and the use of fluorophores with very high extinction coefficients, large quantum yields, and photochemical sta...

show abstract

Section: Discussionmentioning

confidence: 52%

mentioning

confidence: 99%

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline

Best

Merchant

Gopich

et al. 2007

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

208

297

View full text Add to dashboard Cite

show abstract

“…Single molecule fluorescence has proven to be extremely powerful for studying the biophysics of complex biological systems, such as folding of proteins and RNA (22)(23)(24)(25)(26), biomolecular diffusion on membranes and cells (27)(28)(29)(30), protein-protein interactions (31), and protein conformational dynamics (32)(33)(34). In this study, we used single molecule FRET (smFRET) (35,36) to probe the interdomain conformational dynamics of cpSRP43 and how they are affected by binding cpSRP54.…”

mentioning

confidence: 99%

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Gao

Kight

Henderson

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:Targeting of proteins requires a signal recognition particle (SRP) and multiple protein interactions. Results: We observed a decrease in the structural dynamics of cpSRP43 and an increase in substrate affinity upon its binding to cpSRP54. Conclusion: Changes in domain dynamics induced by cpSRP subunit interactions mediate substrate affinity. Significance: Relating structure and dynamics of SRP proteins allows for a better understanding of vectorial targeting within cells.

show abstract

“…Singlemolecule spectroscopy has the inherent ability to separate the signals from subpopulations in heterogeneous mixtures and equilibria, which makes it ideally suited to analyze protein folding reactions (11,12). Specifically, by using single-molecule Förster resonance energy transfer (FRET), intramolecular distances of the unfolded state can be measured even in the presence of a majority of folded molecules (13,14).…”

mentioning

confidence: 99%

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Hoffmann

Kane

Nettels

et al. 2007

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

215

338

View full text Add to dashboard Cite

We have used the combination of single-molecule Fö rster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Fö rster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a ␤-structure content of the collapsed unfolded state of Ϸ20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.Gaussian chain ͉ microfluidic mixing ͉ protein folding ͉ random coil ͉ secondary structure W ith the discovery of small proteins that fold in the absence of populated intermediates (1), our quantitative understanding of the elementary properties of protein folding reactions has made significant advances, including the structural characterization of transition states for folding (2) and the prediction of folding rates from native structure (3-5). One of the most severe limitations for the further development of these approaches is our ignorance about the energetic or structural properties of unfolded † † states of proteins. Because of the structural heterogeneity and complexity of the ensembles of conformations populated by unfolded proteins, their experimental characterization has proven extremely difficult. Traditional methods, such as small-angle scattering techniques (6), provide only global physical properties, e.g., the radius of gyration. In some cases, more detailed structural information can be obtained from NMR (7-10), but these studies usually provide information about the denatured state only under nonnative conditions, typically in the presence of large concentrations of denaturant, or through severe destabilization of the native state induced by covalent modification or mutations. The most interesting and physiologically relevant situation, however, is that of an unfolded sta...

show abstract

Folding dynamics of single GCN-4 peptides by fluorescence resonant energy transfer confocal microscopy

Cited by 153 publications

References 34 publications

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Contact Info

Product

Resources

About