Richard J. Marsh

Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66–86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.

Regulation of 3-Phosphoinositide-Dependent Protein Kinase 1 Activity by Homodimerization in Live Cells

Spatial and temporal regulation of the homodimerization of PDK1 modulates its activity. Sticking Together The “master kinase” phosphoinositide-dependent kinase 1 (PDK1) plays a central role in such processes as cellular proliferation and survival and has a wide range of targets, including protein kinase B (PKB). PDK1 is downstream of phosphatidylinositol 3-kinase (PI3K), and the generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) triggers the translocation of PDK1 and PKB to the plasma membrane, where PDK1 phosphorylates PKB. Although the mechanisms by which PDK1 activates its substrates are well studied, less is known about how the activity of PDK1 is regulated. Masters et al. used a combination of Förster resonance energy transfer (FRET)–based analysis of fluorescently tagged proteins in live cells, as well as computational modeling, to show that a subset of cytosolic PDK1 exists in a homodimeric form. Disruption of the homodimeric interface increased the association between PDK1 and PKB, and this and other evidence suggested that monomeric—rather than dimeric—PDK1 was the active form. Together, these data suggest that homodimerization of PDK1 regulates its activity. 3-Phosphoinositide–dependent kinase 1 (PDK1) plays a central role in regulating the activity of protein kinases that are essential for signaling; however, how PDK1 itself is regulated is largely unknown. We found that homodimerization of PDK1 is a spatially and temporally regulated mechanism for controlling PDK1 activity. We used Förster resonance energy transfer monitored by fluorescence lifetime imaging microscopy to observe PDK1 homodimerization in live cells. A pleckstrin homology (PH) domain–dependent, basal dimeric association of PDK1 was increased upon cell stimulation with growth factors; this association was prevented by a phosphatidylinositol 3-kinase inhibitor and by a mutation in, or a complete deletion of, the PH domain of PDK1. The distinct spatial distribution of PDK1 homodimers relative to that of heterodimers of PDK1 and protein kinase B (PKB), and the ability of monomeric mutants of PDK1 to phosphorylate PKB, suggested that the monomer was the active conformation. Mutation of the autophosphorylation residue threonine-513 to glutamate, which was predicted to destabilize the homodimer interface, enhanced the interaction between PDK1 and PKB and the activity of PKB. Through in vitro, time-resolved fluorescence intensity and anisotropy measurements, combined with existing crystal structures and computational molecular modeling, we determined the geometrical arrangement of the PDK1 homodimer. With this approach, we calculated the size of the population of PDK1 dimers in cells. This description of a previously uncharacterized regulatory mechanism for the activation of PDK1 offers possibilities for controlling PDK1 activity therapeutically.

Stimulated emission depletion of two-photon excited states

Abstract Stimulated emission depletion of fluorescence (STED) from a two-photon excited molecular population is demonstrated for fluorescein in ethylene glycol and methanol. Time resolved fluorescence intensity and anisotropy measurements were made using picosecond time-correlated single photon counting (TCSPC). Pump–dump time delayed total fluorescence intensity measurements were used to characterise the response of the system and to provide additional data on the dump transition dynamics. Cross-sections for the stimulated transition in methanol and ethylene glycol were 1.4 and (3.6±1.0)×10 −16 cm 2 , respectively, the corresponding ground state vibrational lifetimes were 636 and 717±99 fs.

Quantum state-resolved energy redistribution in gas ensembles containing highly excited N2.

A computational model is used to quantify the evolution of quantum state populations as highly vibrationally excited (14)N(2) ((14)N(2)∗) equilibrates in various bath gases. Multicollision energy disposal follows general principles established in related single collision processes. Thus when state-to-state routes permit, maximum amounts of energy are deposited into partner species by direct vibration-to-vibration (V-V) exchange. When these pathways are absent, e.g., when Ar is the bath species, relaxation is very slow and multistaged. Conversely, in a bath of v = 0 (14)N(2) molecules, 16 vibrational quanta (Δv = ± 8) are resonantly exchanged from (v;j) = (8;10) with vibrational equilibration so rapid that rotation and translation still lag far behind after 1000 collisions. Near-resonant V-V exchange dominates the initial phase when (15)N(2) forms the bath gas and although some rotational warming occurs, vibrational modes remain decoupled from, and significantly hotter than, the low heat capacity modes. These forms of behavior seem likely to characterize excited and bath species that have closely similar vibration and rotation constants. More generic in nature is (14)N(2) in O(2) or in a mixture that closely resembles air. Here, asymmetric V-V exchange is a dominant early feature in ensemble evolution but energy differences in the key vibration and rotation quanta lead to V-V energy defects that are compensated for by the low energy modes. This results in much more rapid ensemble equilibration, generally within 400-500 collisions, when O(2) is present even as a minor constituent. Our results are in good general agreement with those obtained from experimental studies of N(2) plasmas both in terms of modal temperatures and initial (first collision cycle) cross-sections.

Restricted State Selection in Fluorescent Protein Forster Resonance Energy Transfer

The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.

Equilibration of vibrationally excited OH in atomic and diatomic bath gases.

In this work, a computational model of state-to-state energy flow in gas ensembles is used to investigate collisional relaxation of excited OH, present as a minor species in various bath gases. Rovibrational quantum state populations are computed for each component species in ensembles consisting of 8000 molecules undergoing cycles of binary collisions. Results are presented as quantum state populations and as (approximate) modal temperatures for each species after each collision cycle. Equilibration of OH is slow with Ar as the partner but much faster when N(2) and/or O(2) forms the bath gas. This accelerated thermalization is shown to be the result of near-resonant vibration-vibration transfer, with vibrational de-excitation in OH matched in energy by excitation in bath molecules. Successive near-resonant events result in an energy cascade. Such processes are highly dependent on molecule pair and on initial OH vibrational state. OH rotational temperatures initially increase, but at equilibration, they are lower than those of other modes. Possible reasons for this observation in molecules such as OH are suggested. There are indications of an order of precedent in the equilibration process, with vibrations taking priority over rotations, and potential explanations for this phenomenon are discussed.

Modeling disequilibrium in gas ensembles: How quantum state populations evolve under multicollision conditions; CO∗+Ar, CO, O2, and N2

The method of Marsh and McCaffery [J. Chem. Phys. 117, 503 (2002)] is used to quantify how rovibrational populations and mode temperatures change as an ensemble of CO molecules, initially excited to (v;j)=(8;12), evolves to thermal equilibrium in a bath gas. The bath gases considered are Ar, N(2), O(2), and CO all at 300 K with the diatomics in their (0;8) rovibrational states. Ensembles generally contain 1000 molecules, 10% of which are excited CO (CO( *)) molecules. State (v;j) populations and mode temperatures of CO* and bath molecules are calculated for successive collisions to 1000 or more. We find that relaxation to local thermodynamic equilibrium occurs in distinct phases that vary widely in rate of cooling. There is especially fast vibration-vibration (VV) exchange in CO*-CO mixtures that is largely decoupled from rotation and translation. Several aspects of ensemble behavior may be rationalized using concepts established in quantum state resolved single collision studies. We demonstrate the existence of a simultaneous energy quasiresonant, angular momentum conserving, low Deltaj VV process that can cause either ultrafast relaxation or up pumping of the kind seen in a number of experiments.

Atom-molecule collisions; the role of nuclear dynamics

Experimental evidence indicates that the physics of molecular collisions is characterised by an underlying simplicity when viewed from the perspective of the dynamics of the nuclei in a particle representation of the species involved. We review this evidence and describe a simple, transparent kinematic theory of inelastic collisions from which the quantum state-resolved outcome of atom-molecule and molecule-molecule collisions are predicted. The principal mechanism of change is linear-to-angular momentum conversion via a torque-arm of molecular dimension constrained to operate within boundaries set by the quantisation of molecular eigenstates and by overall energy conservation. The mechanism is unchanged throughout the wide variety of processes molecules undergo but is modified in a process- and system-specific manner by boundary condition changes and this gives the wide variety of outcomes seen experimentally. The mechanism and boundary conditions may be represented in velocity (momentum)Ðangular momentum diagrams that illustrate vividly the interplay of momentum and energetic factors. Rapid, accurate calculation routines based on these principles reproduce rotational and vibrational distributions observed in experiment over the wide range of inelastic processes that diatomic molecules undergo as well as in atom-diatom reactive encounters.

Low power super resolution fluorescence microscopy by lifetime modification and image reconstruction

We demonstrate a new method for obtaining sub-diffraction resolution in fluorescence microscopy. The technique involves the analysis of the time evolution of fluorescence images in the presence of weak and unstructured (fundamental Gaussian) continuous wave stimulated emission depletion. A reduced point spread functions (PSF) is obtained by the recombination of time segments of the evolving image. A significant reduction in the PSF for 20 nm fluorescent beads (ca. 240 nm to 125 nm) is obtained with an on-sample power of 7.5 mW (17 MW/cm2) - substantially lower than that required for spatially structured stimulated emission depletion microscopy.

Stimulated Emission Depletion and Fluorescence Correlation Spectroscopy of a Branched Quadrupolar Chromophore.

Stimulated emission depletion (STED) and single molecule fluorescence correlation spectroscopy (FCS) are used to determine stimulated emission cross-sections and investigate non-radiative relaxation in a branched quadrupolar chromophore (OM77). The results are used as inputs to simulations of single molecule STED by which the feasibility of STED control of the single molecule fluorescence cycle can be assessed. Single molecule STED in OM77 is shown to be readily achievable; however its effectiveness in reducing triplet trapping is apparently mediated by fast non-radiative relaxation processes other than intersystem crossing and rapid quenching of the triplet state in a non-deoxygenated environment.