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1.
Proc Natl Acad Sci U S A ; 119(12): e2116736119, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35290118

RESUMO

Polymorphism in the structure of amyloid fibrils suggests the existence of many different assembly pathways. Characterization of this heterogeneity is the key to understanding the aggregation mechanism and toxicity, but in practice it is extremely difficult to probe individual aggregation pathways in a mixture. Here, we present development of a method combining single-molecule fluorescence lifetime imaging and deep learning for monitoring individual fibril formation in real time and their high-throughput analysis. A deep neural network (FNet) separates an image of highly overlapping fibrils into single fibril images, which allows for tracking the growth and changes in characteristics of individual fibrils. Using this method, we investigated aggregation of the 42-residue amyloid-ß peptide (Aß42). We demonstrate that highly heterogeneous fibril formation can be quantitatively characterized in terms of the number of cross-ß subunits, elongation speed, growth polarity, and conformation of fibrils. Tracking individual fibril formation and growth also leads to the discovery of a general nucleation mechanism (termed heterogeneous secondary nucleation), where a fibril is formed on the surface of an oligomer with a different structure. Our development will be broadly applicable to characterization of heterogeneous aggregation processes of other proteins.


Assuntos
Peptídeos beta-Amiloides , Aprendizado Profundo , Amiloide/química , Peptídeos beta-Amiloides/metabolismo , Imagem Óptica , Fragmentos de Peptídeos/metabolismo
2.
J Am Chem Soc ; 146(35): 24426-24439, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39177153

RESUMO

The two most abundant isoforms of amyloid-ß (Aß) are the 40- (Aß40) and 42-residue (Aß42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aß aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aß42 and Aß40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aß42 with a small amount of Aß40, while the second phase results mostly from aggregation of Aß40. We also found that the aggregation of Aß42 is slowed by Aß40 while the aggregation of Aß40 is accelerated by Aß42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aß42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aß40 in the co-oligomers in a 1:1 mixture of Aß42 and Aß40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aß42 and Aß40 (1:10), suggesting the critical role of Aß40 in oligomer formation and aggregation.


Assuntos
Peptídeos beta-Amiloides , Transferência Ressonante de Energia de Fluorescência , Fragmentos de Peptídeos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos , Humanos , Imagem Individual de Molécula
3.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34404723

RESUMO

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τc The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Desdobramento de Proteína , Solventes/química , Domínios de Homologia de src , Animais , Drosophila melanogaster , Modelos Moleculares , Termodinâmica
4.
J Chem Phys ; 159(2)2023 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-37431909

RESUMO

In single-molecule free diffusion experiments, molecules spend most of the time outside a laser spot and generate bursts of photons when they diffuse through the focal spot. Only these bursts contain meaningful information and, therefore, are selected using physically reasonable criteria. The analysis of the bursts must take into account the precise way they were chosen. We present new methods that allow one to accurately determine the brightness and diffusivity of individual molecule species from the photon arrival times of selected bursts. We derive analytical expressions for the distribution of inter-photon times (with and without burst selection), the distribution of the number of photons in a burst, and the distribution of photons in a burst with recorded arrival times. The theory accurately treats the bias introduced due to the burst selection criteria. We use a Maximum Likelihood (ML) method to find the molecule's photon count rate and diffusion coefficient from three kinds of data, i.e., the bursts of photons with recorded arrival times (burstML), inter-photon times in bursts (iptML), and the numbers of photon counts in a burst (pcML). The performance of these new methods is tested on simulated photon trajectories and on an experimental system, the fluorophore Atto 488.

5.
Proc Natl Acad Sci U S A ; 115(51): E11924-E11932, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30509980

RESUMO

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer's disease by mitigating against intracellular ß-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-ß(1-42) (Aß42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aß42 with GroEL using a variety of biophysical techniques. Aß42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high-molecular-weight polydisperse aggregates present in fresh soluble Aß42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aß42 reveals the presence of three anchor-binding regions (residues 16-21, 31-34, and 40-41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aß42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aß42 construct, indicating that Aß42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aß42 species and the appearance of Aß42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aß42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Chaperonina 60/antagonistas & inibidores , Chaperonina 60/metabolismo , Síndromes Neurotóxicas/metabolismo , Fragmentos de Peptídeos/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Chaperonina 60/uso terapêutico , Escherichia coli/genética , Escherichia coli/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Microscopia Eletrônica , Modelos Moleculares , Células-Tronco Neurais/efeitos dos fármacos , Síndromes Neurotóxicas/tratamento farmacológico , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Coloração e Rotulagem
6.
Proc Natl Acad Sci U S A ; 114(33): E6812-E6821, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28760960

RESUMO

We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Domínios Proteicos , Multimerização Proteica , Proteína Supressora de Tumor p53/química , Algoritmos , Sequência de Aminoácidos , Carbocianinas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Cinética , Maleimidas/química , Modelos Moleculares , Conformação Proteica , Succinimidas/química
7.
Biophys J ; 117(8): 1456-1466, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31587829

RESUMO

The modern energy landscape theory of protein folding predicts multiple folding pathways connecting a myriad of unfolded conformations and a well-defined folded state. However, direct experimental observation of heterogeneous folding pathways is difficult. Naturally evolved proteins typically exhibit a smooth folding energy landscape for fast and efficient folding by avoiding unfavorable kinetic traps. In this case, rapid fluctuations between unfolded conformations result in apparent two-state behavior and make different pathways indistinguishable. However, the landscape roughness can be different, depending on the selection pressures during evolution. Here, we characterize the unusually rugged folding energy landscape of human immunodeficiency virus-1 protease monomer using single-molecule Förster resonance energy transfer spectroscopy. Our data show that fluctuations between unfolded conformations are slow, which enables the experimental observation of heterogeneous folding pathways as predicted by the landscape theory. Although the landscape ruggedness is sensitive to the mutations and fluorophore locations, the folding rate is similar for various protease constructs. The natural evolution of the protease to have a rugged energy landscape likely results from intrinsic pressures to maintain robust folding when human immunodeficiency virus-1 mutates frequently, which is essential for its survival.


Assuntos
Protease de HIV/química , Simulação de Dinâmica Molecular , Dobramento de Proteína , Transferência Ressonante de Energia de Fluorescência , Protease de HIV/genética , Mutação
8.
Nature ; 502(7473): 685-8, 2013 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-24153185

RESUMO

Kramers developed the theory on how chemical reaction rates are influenced by the viscosity of the medium. At the viscosity of water, the kinetics of unimolecular reactions are described by diffusion of a Brownian particle over a free-energy barrier separating reactants and products. For reactions in solution this famous theory extended Eyring's transition state theory, and is widely applied in physics, chemistry and biology, including to reactions as complex as protein folding. Because the diffusion coefficient of Kramers' theory is determined by the dynamics in the sparsely populated region of the barrier top, its properties have not been directly measured for any molecular system. Here we show that the Kramers diffusion coefficient and free-energy barrier can be characterized by measuring the temperature- and viscosity-dependence of the transition path time for protein folding. The transition path is the small fraction of an equilibrium trajectory for a single molecule when the free-energy barrier separating two states is actually crossed. Its duration, the transition path time, can now be determined from photon trajectories for single protein molecules undergoing folding/unfolding transitions. Our finding of a long transition path time with an unusually small solvent viscosity dependence suggests that internal friction as well as solvent friction determine the Kramers diffusion coefficient for α-helical proteins, as opposed to a breakdown of his theory, which occurs for many small-molecule reactions. It is noteworthy that the new and fundamental information concerning Kramers' theory and the dynamics of barrier crossings obtained here come from experiments on a protein rather than a much simpler chemical or physical system.


Assuntos
Corantes Fluorescentes/análise , Modelos Químicos , Sondas Moleculares/análise , Proteínas/química , Difusão , Transferência Ressonante de Energia de Fluorescência , Fricção , Simulação de Dinâmica Molecular , Fótons , Dobramento de Proteína , Desdobramento de Proteína , Solventes/química , Temperatura , Termodinâmica , Viscosidade
9.
Biophys J ; 114(4): 870-884, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29490247

RESUMO

Monomers of amyloid-ß (Aß) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aß isoforms with 40 (Aß40) and 42 residues (Aß42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ∼35 ns, similar to that of disordered proteins. These results suggest that both Aß40 and Aß42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies.


Assuntos
Peptídeos beta-Amiloides/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Imagem Individual de Molécula/métodos , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Conformação Proteica
10.
Phys Rev Lett ; 115(1): 018101, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-26182121

RESUMO

Although Kramers' theory for diffusive barrier crossing on a 1D free energy profile plays a central role in landscape theory for complex biomolecular processes, it has not yet been rigorously tested by experiment. Here we test this 1D diffusion scenario with single molecule fluorescence measurements of DNA hairpin folding. We find an upper bound of 2.5 µs for the average transition path time, consistent with the predictions by theory with parameters determined from optical tweezer measurements.


Assuntos
DNA de Cadeia Simples/química , Substâncias Macromoleculares/química , Modelos Químicos , Espectrometria de Fluorescência/métodos , Difusão , Conformação de Ácido Nucleico , Pinças Ópticas , Termodinâmica
11.
Phys Chem Chem Phys ; 16(35): 18644-57, 2014 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-25088495

RESUMO

Single-molecule spectroscopy is widely used to study macromolecular dynamics. Although this technique provides unique information that cannot be obtained at the ensemble level, the possibility of studying fast molecular dynamics is limited by the number of photons detected per unit time (photon count rate), which is proportional to the illumination intensity. However, simply increasing the illumination intensity often does not help because of various photophysical and photochemical problems. In this Perspective, we show how to improve the dynamic range of single-molecule fluorescence spectroscopy at a given photon count rate by considering each and every photon and using a maximum likelihood method. For a photon trajectory with recorded photon colors and inter-photon times, the parameters of a model describing molecular dynamics are obtained by maximizing the appropriate likelihood function. We discuss various likelihood functions, their applicability, and the accuracy of the extracted parameters. The maximum likelihood method has been applied to analyze the experiments on fast two-state protein folding and to measure transition path times. Utilizing other information such as fluorescence lifetimes is discussed in the framework of two-dimensional FRET efficiency-lifetime histograms.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Modelos Teóricos , Cinética , Simulação de Dinâmica Molecular , Fótons , Dobramento de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteínas/química
12.
Proc Natl Acad Sci U S A ; 108(23): 9437-42, 2011 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-21596998

RESUMO

ESCRT-I is required for the sorting of integral membrane proteins to the lysosome, or vacuole in yeast, for cytokinesis in animal cells, and for the budding of HIV-1 from human macrophages and T lymphocytes. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37, and Mvb12. The crystal structures of the core complex and the ubiquitin E2 variant and Vps28 C-terminal domains have been determined, but internal flexibility has prevented crystallization of intact ESCRT-I. Here we have characterized the structure of ESCRT-I in solution by simultaneous structural refinement against small-angle X-ray scattering and double electron-electron resonance spectroscopy of spin-labeled complexes. An ensemble of at least six structures, comprising an equally populated mixture of closed and open conformations, was necessary to fit all of the data. This structural ensemble was cross-validated against single-molecule FRET spectroscopy, which suggested the presence of a continuum of open states. ESCRT-I in solution thus appears to consist of an approximately 50% population of one or a few related closed conformations, with the other 50% populating a continuum of open conformations. These conformations provide reference points for the structural pathway by which ESCRT-I induces membrane buds.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Saccharomyces cerevisiae/química , Difração de Raios X/métodos , Algoritmos , Anisotropia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espalhamento a Baixo Ângulo , Soluções
13.
J Phys Chem B ; 128(23): 5576-5589, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38833567

RESUMO

Single-molecule free diffusion experiments enable accurate quantification of coexisting species or states. However, unequal brightness and diffusivity introduce a burst selection bias and affect the interpretation of experimental results. We address this issue with a photon-by-photon maximum likelihood method, burstML, which explicitly considers burst selection criteria. BurstML accurately estimates parameters, including photon count rates, diffusion times, Förster resonance energy transfer (FRET) efficiencies, and population, even in cases where species are poorly distinguished in FRET efficiency histograms. We develop a quantitative theory that determines the fraction of photon bursts corresponding to each species and thus obtain accurate species populations from the measured burst fractions. In addition, we provide a simple approximate formula for burst fractions and establish the range of parameters where unequal brightness and diffusivity can significantly affect the results obtained by conventional methods. The performance of the burstML method is compared with that of a maximum likelihood method that assumes equal species brightness and diffusivity, as well as standard Gaussian fitting of FRET efficiency histograms, using both simulated and real single-molecule data for cold-shock protein, protein L, and protein G. The burstML method enhances the accuracy of parameter estimation in single-molecule fluorescence studies.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Difusão , Fótons , Funções Verossimilhança , Imagem Individual de Molécula/métodos
14.
Chem Phys ; 422: 229-237, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-24443626

RESUMO

Folding and unfolding rates for the ultrafast folding villin subdomain were determined from a photon-by-photon analysis of fluorescence trajectories in single molecule FRET experiments. One of the obstacles to measuring fast kinetics in single molecule fluorescence experiments is blinking of the fluorophores on a timescale that is not well separated from the process of interest. By incorporating acceptor blinking into a two-state kinetics model, we show that it is possible to extract accurate rate coefficients on the microsecond time scale for folding and unfolding using the maximum likelihood method of I.V. Gopich and A. Szabo. This method yields the most likely parameters of a given model that can reproduce the observed photon trajectories. The extracted parameters agree with both the decay rate of the donor-acceptor cross correlation function and the results of ensemble equilibrium and kinetic experiments using nanosecond laser temperature jump.

15.
PNAS Nexus ; 2(8): pgad253, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37564361

RESUMO

Soluble oligomers produced during protein aggregation have been thought to be toxic species causing various diseases. Characterization of these oligomers is difficult because oligomers are a heterogeneous mixture, which is not readily separable, and may appear transiently during aggregation. Single-molecule spectroscopy can provide valuable information by detecting individual oligomers, but there have been various problems in determining the size and concentration of oligomers. In this work, we develop and use a method that analyzes single-molecule fluorescence burst data of freely diffusing molecules in solution based on molecular diffusion theory and maximum likelihood method. We demonstrate that the photon count rate, diffusion time, population, and Förster resonance energy transfer (FRET) efficiency can be accurately determined from simulated data and the experimental data of a known oligomerization system, the tetramerization domain of p53. We used this method to characterize the oligomers of the 42-residue amyloid-ß (Aß42) peptide. Combining peptide incubation in a plate reader and single-molecule free-diffusion experiments allows for the detection of stable oligomers appearing at various stages of aggregation. We find that the average size of these oligomers is 70-mer and their overall population is very low, less than 1 nM, in the early and middle stages of aggregation of 1 µM Aß42 peptide. Based on their average size and long diffusion time, we predict the oligomers have a highly elongated rod-like shape.

16.
Nat Commun ; 14(1): 5438, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37673863

RESUMO

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.


Assuntos
Dobramento de RNA , Riboswitch , Espalhamento a Baixo Ângulo , Difração de Raios X , RNA , Riboswitch/genética , Aminoácidos , Chaperonas Moleculares
17.
Proc Natl Acad Sci U S A ; 106(29): 11837-44, 2009 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-19584244

RESUMO

Transition paths are a uniquely single-molecule property not yet observed for any molecular process in solution. The duration of transition paths is the tiny fraction of the time in an equilibrium single-molecule trajectory when the process actually happens. Here, we report the determination of an upper bound for the transition path time for protein folding from photon-by-photon trajectories. FRET trajectories were measured on single molecules of the dye-labeled, 56-residue 2-state protein GB1, immobilized on a glass surface via a biotin-streptavidin-biotin linkage. Characterization of individual emitted photons by their wavelength, polarization, and absolute and relative time of arrival after picosecond excitation allowed the determination of distributions of FRET efficiencies, donor and acceptor lifetimes, steady state polarizations, and waiting times in the folded and unfolded states. Comparison with the results for freely diffusing molecules showed that immobilization has no detectable effect on the structure or dynamics of the unfolded protein and only a small effect on the folding/unfolding kinetics. Analysis of the photon-by-photon trajectories yields a transition path time <200 micros, >10,000 times shorter than the mean waiting time in the unfolded state (the inverse of the folding rate coefficient). Szabo's theory for diffusive transition paths shows that this upper bound for the transition path time is consistent with previous estimates of the Kramers preexponential factor for the rate coefficient, and predicts that the transition path time is remarkably insensitive to the folding rate, with only a 2-fold difference for rate coefficients that differ by 10(5)-fold.


Assuntos
Fótons , Dobramento de Proteína , Biotina/metabolismo , Corantes , Transferência Ressonante de Energia de Fluorescência , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Luz , Fenômenos Físicos , Dobramento de Proteína/efeitos dos fármacos , Estreptavidina/metabolismo , Fatores de Tempo , Ureia/farmacologia
18.
Methods Mol Biol ; 2376: 247-282, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34845614

RESUMO

Inter-dye distances and conformational dynamics can be studied using single-molecule FRET measurements. We consider two approaches to analyze sequences of photons with recorded photon colors and arrival times. The first approach is based on FRET efficiency histograms obtained from binned photon sequences. The experimental histograms are compared with the theoretical histograms obtained using the joint distribution of acceptor and donor photons or the Gaussian approximation. In the second approach, a photon sequence is analyzed without binning. The parameters of a model describing conformational dynamics are found by maximizing the appropriate likelihood function. The first approach is simpler, while the second one is more accurate, especially when the population of species is small and transition rates are fast. The likelihood-based analysis as well as the recoloring method has the advantage that diffusion of molecules through the laser focus can be rigorously handled.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Difusão , Funções Verossimilhança , Conformação Molecular , Fótons
19.
J Phys Chem A ; 115(16): 3642-56, 2011 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-20509636

RESUMO

Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (<10%) population of the minor component where the cross-correlation function was too noisy to obtain any useful information. The rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded molecules are present, this method yields rate coefficients in very good agreement with those obtained with the maximum likelihood method. As a first step toward characterizing transition paths, the Viterbi algorithm was used to locate the most probable transition points in the photon trajectories.


Assuntos
Fótons , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Algoritmos , Biotina/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Modelos Moleculares , Proteínas/síntese química , Proteínas/isolamento & purificação , Estreptavidina/química
20.
Biophys J ; 98(4): 696-706, 2010 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-20159166

RESUMO

Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity.


Assuntos
Corantes/química , Transferência Ressonante de Energia de Fluorescência , Proteínas/metabolismo , Sequência de Aminoácidos , Difusão , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Proteínas/química
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