Alexander Kyrychenko

High-performance time-resolved fluorescence by direct waveform recording.

We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 10(5) times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats.

Conformational switching of the diphtheria toxin T domain.

The diphtheria toxin T domain translocates the catalytic C domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited wild-type-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by the H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.

Kinetic Intermediate Reveals Staggered pH-Dependent Transitions along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain

The pH-triggered membrane insertion pathway of the T-domain of diphtheria toxin was studied using site-selective fluorescence labeling with subsequent application of several spectroscopic techniques (e.g., fluorescence correlation spectroscopy, FRET, lifetime quenching, and kinetic fluorescence). FCS measurements indicate that pH-dependent formation of the membrane-competent form depends only slightly on the amount of anionic lipids in the membrane. The subsequent transbilayer insertion, however, is strongly favored by anionic lipids. Kinetic FRET measurements between the donor-labeled T-domain and acceptor-labeled lipid vesicles demonstrate rapid membrane association at all pH values for which binding occurs. In contrast, the transmembrane insertion kinetics is significantly slower and is also both pH- and lipid-dependent. Analysis of kinetic behavior of binding and insertion indicates the presence of several interfacial intermediates on the insertion pathway of the T-domain, from soluble W-state to transmembrane T-state. Intermediate interfacial I-state can be trapped in membranes with low content of anionic lipids (10%). In membranes of greater anionic lipid content, another pH-dependent transition results in the formation of the insertion-competent state and subsequent transmembrane insertion. Comparison of the results of various kinetic and equilibrium experiments suggests that the pH dependences determining membrane association and transbilayer insertion transitions are different but staggered. Anionic lipids not only assist in formation of the insertion-competent form but also lower the kinetic barrier for the final insertion.

pH-triggered conformational switching of the diphtheria toxin T-domain: the roles of N-terminal histidines.

pH-induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side-chain protonation accompanying the formation of the membrane-competent state of the toxins translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry [e.g., long-microsecond-range, all-atom molecular dynamics (MD) simulations; continuum electrostatics calculations; and thermodynamic integration (TI)] with several experimental techniques of fluorescence spectroscopy. TI calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mol), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over 8 μs demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2 and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.

Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662-683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning.

Thermodynamic measurements of bilayer insertion of a single transmembrane helix chaperoned by fluorinated surfactants.

Accurate determination of the free energy of transfer of a helical segment from an aqueous into a transmembrane (TM) conformation is essential for understanding and predicting the folding and stability of membrane proteins. Until recently, direct thermodynamically sound measurements of free energy of insertion of hydrophobic TM peptides were impossible due to peptide aggregation outside the lipid bilayer. Here, we overcome this problem by using fluorinated surfactants that are capable of preventing aggregation but, unlike detergents, do not themselves interact with the bilayer. We have applied the fluorescence correlation spectroscopy methodology to study surfactant-chaperoned insertion into preformed POPC (palmitoyloleoylphosphatidylcholine) vesicles of the two well-studied dye-labeled TM peptides of different lengths: WALP23 and WALP27. Extrapolation of the apparent free-energy values measured in the presence of surfactants to a zero surfactant concentration yielded free-energy values of -9.0±0.1 and -10.0±0.1 kcal/mol for insertion of WALP23 and WALP27, respectively. Circular dichroism measurements confirmed helical structure of peptides in lipid bilayer, in the presence of surfactants, and in aqueous mixtures of organic solvents. From a combination of thermodynamic and conformational measurements, we conclude that the partitioning of a four-residue L-A-L-A segment in the context of a continuous helical conformation from an aqueous environment into the hydrocarbon core of the membrane has a favorable free energy of 1 kcal/mol. Our measurements, combined with the predictions of two independent experimental hydrophobicity scales, indicate that the per-residue cost of transfer of the helical backbone from water to the hydrocarbon core of the lipid bilayer is unfavorable and is equal to +2.13±0.17 kcal/mol.

Lipid Headgroups Modulate Membrane Insertion of pHLIP Peptide

The pH low insertion peptide (pHLIP) is an important tool for drug delivery and visualization of acidic tissues produced by various maladies, including cancer, inflammation, and ischemia. Numerous studies indicate that pHLIP exists in three states: unfolded and soluble in water at neutral pH (State I), unfolded and bound to the surface of a phosphatidylcholine membrane at neutral pH (State II), and inserted across the membrane as an α-helix at low pH (State III). Here we report how changes in lipid composition modulate this insertion scheme. First, the presence of either anionic lipids, cholesterol, or phosphoethanolamine eliminates membrane binding at neutral pH (State II). Second, the apparent pKa for the insertion transition (State I → State III) is increased with increasing content of anionic lipids, suggesting that electrostatic interactions in the interfacial region modulate protonation of acidic residues of pHLIP responsible for transbilayer insertion. These findings indicate a possibility for triggering protonation-coupled conformational switching in proteins at membrane interfaces through changes in lipid composition.

STEADY-STATE AND TIME-RESOLVED FLUORESCENCE QUENCHING WITH TRANSITION METAL IONS AS SHORT-DISTANCE PROBES FOR PROTEIN CONFORMATION

A series of model dye-labeled histidine-containing peptides was used to investigate the nature of the quenching mechanism with Cu(2+) and Ni(2+). The strong reduction in steady-state fluorescence was found to be unaccompanied by any noticeable changes in lifetime kinetics. This static nature of quenching is not consistent with the dynamic Förster resonance energy transfer (FRET) phenomenon, which was assumed to dominate the quenching mechanism, and is likely caused by shorter range orbital coupling. Our results indicate that the FRET-like sixth power of distance dependence of quenching cannot be automatically assumed for transition metal ions and that time-resolved measurements should be used to distinguish various quenching mechanisms.

Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching

Accurate determination of the depth of membrane penetration of a fluorescent probe, attached to a lipid, protein, or other macromolecule of interest, using depth-dependent quenching methodology is complicated by thermal motion in the lipid bilayer. Here, we suggest that a combination of steady-state and time-resolved measurements can be used to generate a static quenching profile that reduces the contribution from transverse diffusion occurring during the excited-state lifetime. This procedure results in narrower quenching profiles, compared with those obtained by traditional measurements, and thus improves precision in determination of the underlying depth distribution of the probe.

Computational refinement of spectroscopic FRET measurements

This article supplies raw data related to a research article entitled “Joint refinement of FRET measurements using spectroscopic and computational tools” (Kyrychenko et al., 2017) [1], in which we demonstrate the use of molecular dynamics simulations to estimate FRET orientational factors in a benchmark donor-linker-acceptor system of enhanced cyan (ECFP) and enhanced yellow (EYFP) fluorescent proteins. This can improve the recalculation of donor-acceptor distance information from single-molecule FRET measurements.