Critical Evaluation of the Two-State Model Describing the Equilibrium Unfolding of the PI3K SH3 Domain by Time-Resolved Fluorescence Resonance Energy Transfer

It appears that equilibrium unfolding transitions of many small proteins can be described as two-state transitions, because the probes commonly used to measure such transitions cannot detect the underlying heterogeneity inherent in protein folding and unfolding reactions. Time-resolved fluorescence or Forster resonance energy transfer (TRFRET) measurements have the potential to uncover such heterogeneity and to test the cooperativity of protein folding reactions. Here, TRFRET measurements have been used to study the equilibrium unfolding of the SH3 domain of PI3 kinase. The single tryptophan residue (W53) was used as the FRET donor, and a covalently attached thionitrobenzoate moiety at either of two sites (C17 and C70) was used as the FRET acceptor. The individual lifetime and amplitude components estimated from fitting the fluorescence decay kinetics to the sum of three or four exponentials were determined over a range of denaturant concentrations. The equilibrium unfolding transitions reported by these components were found to be noncoincident, suggesting the presence of multiple conformations in equilibrium during the course of unfolding. Fluorescence lifetime distributions were also generated by the model-free maximum entropy method of analysis. Different segments of the protein were found to show differences in the expansion of the native state at low denaturant concentrations, suggestive of gradual structural transitions. The unfolded protein was found to swell at increasingly high denaturant concentrations. The evolution of the fluorescence lifetime distributions with increasing denaturant concentration was also found to be incompatible with a two-state equilibrium unfolding model

Photophysics of 2‑(4′-Amino-2′-hydroxyphenyl)‑1<i>H</i>‑imidazo-[4,5‑<i>c</i>]pyridine and Its Analogues: Intramolecular Proton Transfer versus Intramolecular Charge Transfer

Photophysical characteristics of 2-(4′-amino-2′-hydroxyphenyl)-1<i>H</i>-imidazo-[4,5-<i>c</i>]­pyridine (AHPIP-c) have been studied in various aprotic and protic solvents using UV–visible, steady state fluorescence and time-resolved fluorescence spectroscopic techniques. To comprehend the competition between the intramolecular charge transfer (ICT) and the excited state intramolecular proton transfer (ESIPT) processes, the photophysical properties of 2-(4′-amino-2′-methoxyphenyl)-1<i>H</i>-imidazo-[4,5-<i>c</i>]­pyridine (AMPIP-c) and 2-(4′-aminophenyl)-1<i>H</i>-imidazo-[4,5-<i>c</i>]­pyridine (APIP-c) were also investigated. Though APIP-c displays twisted ICT (TICT) emission in protic solvents, AHPIP-c exhibits normal and tautomer emissions in aprotic as well as in protic solvents due to ESIPT. However, the methoxy derivative, AMPIP-c, emits weak TICT fluorescence in methanol

Enhancing Excited State Intramolecular Proton Transfer in 2‑(2′-Hydroxyphenyl)benzimidazole and Its Nitrogen-Substituted Analogues by β‑Cyclodextrin: The Effect of Nitrogen Substitution

Excited state intramolecular proton transfer (ESIPT) in nitrogen-substituted analogues of 2-(2′-hydroxyphenyl)­benzimidazole (HPBI), 2-(2′-hydroxyphenyl)-3<i>H</i>-imidazo­[4,5-<i>b</i>]­pyridine (HPIP-b), and 2-(2′-hydroxyphenyl)-3<i>H</i>-imidazo­[4,5-<i>c</i>]­pyridine (HPIP-c) have been investigated in a β-cyclodextrin (β-CD) nanocavity and compared with that of HPBI. The stoichiometry and the binding constants of the complexes were determined by tautomer emissions. Both p<i>K</i>_a and NMR experiments were employed to determine the orientation of the molecules inside of the β-CD cavity. Huge enhancement in the tautomer emission of HPIP-b and HPIP-c compared to that of HPBI in β-CD suggests that not only is the ESIPT favored inside of the cavity, but also, the environment reduces the nonradiative decay through the formation of an intramolecular charge-transfer (ICT) state. Unlike HPBI, the tautomer emission to normal emission ratio of HPIP-b increases from 0.9 to 2.6, and that of HPIP-c increases from 4.9 to 7.4 in 15 mM β-CD. The effect of dimethylsulfoxide (DMSO) on complexation was also investigated for all three guest molecules. In DMSO, HPBI is present in neutral form, but the nitrogen-substituted analogues are present in both neutral and monoanionic forms. However, in DMSO upon encapsulation by β-CD, all three molecules are present in both neutral and monoanionic forms in the nanocavity. The monoanion is stabilized more inside of the β-CD cavity. The studies revealed that the ESIPT of nitrogen-substituted analogues is more susceptible to the environment than HPBI, and therefore, they are more promising probes

Depth-Dependent Heterogeneity in Membranes by Fluorescence Lifetime Distribution Analysis

Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function

Site-Specific Fluorescence Dynamics of α‑Synuclein Fibrils Using Time-Resolved Fluorescence Studies: Effect of Familial Parkinson’s Disease-Associated Mutations

α-Synuclein (α-Syn) aggregation is directly implicated in both the initiation and spreading of Parkinson’s Diseases (PD) pathogenesis. Although the familial PD-associated mutations (A53T, E46K, and A30P) are known to affect the aggregation kinetics of α-Syn <i>in vitro</i>, their structural differences in resultant fibrils are largely unknown. In this report we studied the site-specific dynamics of wild type (wt) α-Syn and its three PD mutant fibrils using time-resolved fluorescence intensity, anisotropy decay kinetics, and fluorescence quenching. Our data suggest that the N- and C-terminus are more flexible and exposed compared to the middle non-amyloid-β component (NAC) region of wt and PD mutant α-Syn fibrils. Yet the N-terminus showed great conformational heterogeneity compared to the C-terminus for all these proteins. 71 position of E46K showed more flexibility and solvent exposure compared to other α-Syns, whereas both E46K and A53T fibrils possess a more rigid C-terminus compared to wt and A30P. The present data suggest that wt and PD mutant fibrils possess large differences in flexibility and solvent exposure at different positions, which may contribute to their different pathogenicity in PD

Active site dynamics of wild type and 6B lipase.

<p>(A) RMSD of C_α atoms of wild type and 6B lipases from their energy minimized crystal structures as a function of MD simulation time. For clarity, single simulation data is shown for both wild type and 6B lipase while others are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188.s003" target="_blank">Fig. S2A</a>. (B) RMSF of C_α atoms of individual residues during 2–20 ns simulation time. Active site residues positions are shown as solid spheres. (C) Typical time-resolved fluorescence anisotropic decay profiles of acrylodan attached to C77 in wild type and 6B lipase background.</p

Depth-Dependent Membrane Ordering by Hemagglutinin Fusion Peptide Promotes Fusion

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Membrane fusion is essential for the entry of lipid-sheathed viruses such as influenza and HIV. Influenza virus is internalized via receptor-mediated endocytosis and then fuses with the endosomal membrane at low pH. Hemagglutinin, a glycoprotein found on the surface of influenza virus, is responsible for the fusion of the viral sheath with the endosomal membrane. The ∼20 amino acid long N-terminus of hemagglutinin, known as the fusion peptide, plays a crucial role in the viral fusion process. Although there exists vast literature on the importance and role of the fusion peptide in promoting membrane fusion, there is no consensus on the mechanism by which it promotes fusion. A recent report suggested that the fusion peptide occupies and orders space in the outer leaflets of contacting bilayers so as to promote acyl chain protrusion into interbilayer space and promote fusion “stalk” formation. We report here the effect of the wild type, G1S, G1V, and W14A mutants of hemagglutinin fusion peptide on depth-dependent ordering of model membranes along the bilayer normal. We utilized fluorescence anisotropy, lifetime measurements, and lifetime distribution analyses of different anthroyloxy stearic acid probes (<i>n</i>-AS) in order to examine the effect of fusion peptides at various depths along the bilayer normal. Wild type peptide uniquely ordered a region ∼12 Å from the bilayer midpoint, W14A and G1S mutants mainly ordered the bilayer interface, while G1V had little ordering influence. On the basis of recent analysis of the effects of these peptides on fusion, ordering of the mid-upper region of the bilayer appears to promote fusion pore formation, while ordering of the bilayer interface inhibits it

Catalytic parameters of lipases at room temperature (∼20°C).

<p>Catalytic parameters of lipases at room temperature (∼20°C).</p

Active site geometry of wild type and 6B lipase during 2–20 ns MD simulations.

<p>(A) Frequency distribution of MD simulation structural snapshots as a function of distances between hydroxyl oxygen of S77 and imidazole nitrogen of H156. (B) Frequency distribution of MD simulation structural snapshots as a function of distances between imidazole nitrogen of H156 and carboxylate oxygen of D133. (C) Frequency distribution of MD simulation structural snapshots as a function of RMSD of their catalytically important atoms (hydroxyl oxygen of S77, imidazole nitrogens of H156, carboxylate oxygen of D133 and peptidic nitrogens of I12 and M78) to that of transition state analog bound crystal structure (PDB id: 1R4Z, Chain A).</p

Active site in transition state bound and free form.

<p>Structural overlap of active site of the free wild type lipase and in complex with covalently attached transition state analog (chain A of PDB id: 1I6W and 1R4Z). Transition state analog is O-[(R)-1,2-O-isopropylidene-sn-glycerol]6-hexenyl phosphonate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188-Labeikovsky1" target="_blank">[34]</a>. Free enzyme is shown in green while complex is shown in elemental color. Side chains are shown as sticks while backbone as lines. Stereo figure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188.s007" target="_blank">Fig. S6</a>.</p