Fluorescence Energy Transfer Kinetics Research Articles

Probing Melittin Helix−Coil Equilibria in Solutions and Vesicles

Melittin is a toxic, amphipathic peptide that rearranges from a random coil in solution to a helical structure upon binding to cell membranes or lipid vesicles. We have found that mutation of the valine at position five of the peptide to a phenylalanine or 3-nitrotyrosine induces aggregation and helix formation at low concentrations (20-80 microM). Donor-acceptor distances obtained from analyses of fluorescence energy transfer kinetics experiments with the 3-nitrotyrosine mutant indicate that both coil and helix structures are present in 2 and 20 microM aqueous solutions. The helical peptide population increases upon addition of phospholipid vesicles or in high ionic strength solutions.

1P-094 蛍光エネルギー移動キネティクス測定によるACBPフオールディング機構の解明(日本生物物理学会若手奨励賞選考会,若手招待講演,第46回日本生物物理学会年会)

1P-094 蛍光エネルギー移動キネティクス測定によるACBPフオールディング機構の解明(日本生物物理学会若手奨励賞選考会,若手招待講演,第46回日本生物物理学会年会)

Site-specific collapse dynamics guide the formation of the cytochrome c′ four-helix bundle

The evolution of tryptophan-to-heme (W/heme) distance distributions extracted from analysis of fluorescence energy transfer kinetics during the refolding of Rhodopseudomonas palustris cytochrome c' reveals dramatic differences between two variants [W32 (Q1A/F32W/W72F) and W72 (Q1A)]. Both W32/heme and W72/heme distance distributions measured at the earliest time point attainable with a continuous-flow mixer (150 mus) confirm that the polypeptide ensemble is not uniformly collapsed and that native structure is not formed. Time-resolved fluorescence spectra indicate that W32 is sequestered from the aqueous solution during the first 700 mus of folding, whereas W72 remains exposed to solvent. The first moment of the W32/heme distance distribution evolves to its native value faster than that of W72, suggesting that the approach of W32 to the heme precedes that of W72.

Refolding a beta-barrel membrane protein

The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a β-barrel membrane protein. The amphiphilic, β-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus. While the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate β-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate. Fluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.

Snapshots of cytochrome c folding.

Dansyl-to-heme distance distributions [P(r)] during folding have been determined in five variants of Saccharomyces cerevisiae iso-1 ferricytochrome c (labeled at mutant Cys residues 4, 39, 50, 66, and 99) by analysis of fluorescence energy-transfer kinetics. Moment analysis of the P(r) distributions clearly indicates that cytochrome c refolding is not a simple two-state process. After 1 ms of folding, the polypeptide ensemble is not uniformly collapsed and there are site variations in the relative populations of collapsed structures. P(r) distributions reveal structural features of the multiple intermediate species and evolution of the polypeptide ensemble.

α-Synuclein structures from fluorescence energy-transfer kinetics: Implications for the role of the protein in Parkinson's disease

Parkinson's disease is associated with the deposition and accumulation of alpha-synuclein fibrils in the brain. A30P and A53T mutations have been linked to the early-onset familial disease state. Time-resolved tryptophan fluorescence energy-transfer measurements have been used to probe the structures of pseudo-wild-type and mutant (A30P) alpha-synucleins at physiological pH (7.4), in acidic pH (4.4) solutions, and in the presence of SDS micelles, a membrane mimic. Fluorescent donor-energy acceptor (DA) distance distributions for six different tryptophan/3-nitro-tyrosine pairs reveal the presence of compact, intermediate, and extended conformations of the protein. CD spectra indicate that the protein develops substantial helical structure in the presence of SDS micelles. DA distributions show that micelles induce compaction in the N-terminal region and expansion of the acidic C terminus. In acidic solutions, there is an increased population of collapsed structures in the C-terminal region. Energy-transfer measurements demonstrate that the average DA distances for the W4-Y19 and Y19-W39 pairs are longer in one of the two disease-related mutants (A30P).

Conformations of Unfolded S. cerevisiae Cytochrome c Probed by Fluorescence Energy Transfer Kinetics

AbstractEmploying fluorescence energy transfer kinetics, we have shown that substantial populations of compact molecules are present in GuHCl‐denatured CX‐dansyl‐labeled (X = 39, 85, 102) S. cerevisiae iso‐1 cytochrome c. Fully 40% of unfolded molecules are in compact conformations in the C39 protein.

Structural features of the cytochrome C molten globule revealed by fluorescence energy transfer kinetics.

Nonnative states of proteins are involved in a variety of cellular processes, including translocation of proteins across membranes and formation of amyloid fibrils. Probes that report on the structural heterogeneity of a polypeptide ensemble could resolve ambiguities in the classification of these states. Employing fluorescence energy transfer kinetics, we have shown that added anions shift the equilibrium between the compact and extended polypeptide structures that are present during refolding of Saccaromyces cerevisiae iso-1 cytochrome c. Specifically, at high salt concentrations (>/=700 mM), all of the polypeptides are compact with a mean C-terminal fluorophore-heme separation quite close to that in the native protein (25 A).

Structural features of cytochrome c' folding intermediates revealed by fluorescence energy-transfer kinetics.

We employed fluorescence energy-transfer probes to investigate the polypeptide dynamics accompanying cytochrome c' folding. Analysis of fluorescence energy-transfer kinetics from wild-type Trp-72 or Trp-32 in a crystallographically characterized (1.78 A) Q1A/F32W/W72F mutant shows that there is structural heterogeneity in denatured cytochrome c'. Even at guanidine hydrochloride concentrations well beyond the unfolding transition, a substantial fraction of the polypeptides ( approximately 50%) adopts compact conformations (tryptophan-to-heme distance, approximately 25 A) in both pseudo-wild-type (Q1A) and mutant proteins. A burst phase (< or =5 ms) is revealed when stopped flow-triggered refolding is probed by tryptophan intensity: measurements on the Q1A protein show that approximately 75% of the Trp-72 fluorescence (83% for Trp-32) is quenched within the mixing deadtime, suggesting that most of the polypeptides have collapsed.

Using deeply trapped intermediates to map the cytochrome c folding landscape.

Replacement of iron with cobalt(III) selectively introduces a deep trap in the folding-energy landscape of the heme protein cytochrome c. Remarkably, neither the protein structure nor the folding thermodynamics is perturbed by this metal-ion substitution, as shown by data from spectroscopic and x-ray diffraction experiments. Through kinetics measurements, we have found parallel folding pathways involving several different misligated Co(III) species, and, as these folding intermediates persist for several hours under certain conditions, we have been able to elucidate fully their spectroscopic properties. The results, along with an analysis of the fluorescence energy-transfer kinetics during refolding, show that rapidly equilibrating populations of compact and extended polypeptide conformations are present until all molecules have reached the native structure. These measurements provide direct evidence that collapsed denatured structures are not substantially more stable than extended conformations of cytochrome c.

Mapping the Cytochrome c Folding Landscape

The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S.cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations: a collection of collapsed structures with an average fluorophore-heme distance (r) of 27 A and a roughly equal population of extended polypeptides with r > 50 A. Molecules with the native fold appear on a time scale regulated by heme ligation events ( approximately 300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure.

Effective Lattice Behavior of Fluorescence Energy Transfer at Lamellar Macromolecular Interfaces

Fluorescence energy transfer between donors and acceptors confined to macromolecular interfaces is considered. In particular, we discuss two theoretical models for the ensemble-average fluorescence intensity decay of the donor when both fluorophores are incorporated into a planar (e.g., lamellar) interface. The first model is based on a continuous distribution of donor and acceptor molecules on a two-dimensional surface, whereas the other assumes a discrete distribution of fluorophores along the nodes of a two-dimensional square lattice. Results for the discrete model show that the fluorescence intensity kinetics of a donor depends strongly on the geometry of the molecular distribution (i.e., the lattice constant) and the photophysics of fluorophores (i.e., critical radius of the energy transfer). Furthermore, a “discrete molecular distribution” might manifest itself in the experimental data as an increase in the apparent dimensionality of the energy transfer with increasing acceptor concentration. Altogether, the experimental and theoretical underpinnings indicate the enormous potential of using fluorescence energy-transfer kinetics for revealing structural features of molecular ensembles (i.e., geometry, shape) based on a single experimental measurement. However, further understanding the effects of restricted geometries on the fluorescence energy transfer is required to take full advantage of this information. Basic theoretical considerations to that end are provided.