Angus J. Bain

Separating NADH and NADPH fluorescence in live cells and tissues using FLIM

NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles.

Ligand-Dependent Conformational Equilibria of Serum Albumin Revealed by Tryptophan Fluorescence Quenching

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.

Picosecond photoisomerization and rotational reorientation dynamics in solution

The trans–cis isomerization rates for stiff‐diphenylbutadiene (S‐DPB) in n‐alkane solvents were measured using single photon counting methods and the rotational reorientation times τR for S‐DPB and trans stilbene were obtained by picosecond polarization spectroscopy. In neither case did τR vs viscosity show Stokes–Einstein–Debye (SED) behavior. The values of τR were used to calculate the angular velocity correlation frequencies β using the Hubbard relation. The variation of isomerization rate with β was found to be predicted well by the Kramers equation when barrier frequencies of 154 cm−1 for stilbene and 16 cm−1 for S‐DPB were used. This Kramers‐Hubbard fit finesses questions regarding the validity of the one dimensional Kramers model and focuses attention on the SED equation. The dynamical relationship between the torsional friction, important in isomerization, and rotational friction, which determines the overall angular motion of the molecules, is discussed.

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.

Complete determination of the state multipoles of rotationally resolved polarized fluorescence using a single experimental geometry

When laser radiation is used to prepare single rovibronic levels in molecules, the excited state Mj distribution is invariably polarized. In many such experiments the polarization of the excited state is ignored, which is an inadequate basis for accurate work as much valuable detail is lost. A better approach is a completely polarization resolved experiment in which the preparation, dynamics, and detection of the excited state polarization components (the state multipoles JJρKQ) are fully described. A treatment of polarized excitation in terms of the state multipoles JJρKQ is presented and consideration of excited state symmetry indicates that a common experimental geometry for linearly and circularly polarized excitation is feasible. A complete determination of the state multipoles (K=0,1,2) is shown to be possible within a single experimental geometry. It is shown that neglect of polarization phenomena can lead to ambiguities in the interpretation of some experiments.

On the measurement of molecular anisotropies using laser techniques. I. Polarized laser fluorescence

The tensor density matrix formalism is used to derive expressions for the circular and linear polarization of laser‐induced fluorescence from molecules which have an anisotropic distribution in the spatial orientation of their ground state angular momentum components. The generalized anisotropic distribution is expressed as a series of state multipolar moments and it is shown that the excited state multipolar moments created therefrom by the absorption of laser radiation may be quite complex even in the absence of perturbations which cause cross relaxation. Under these circumstances, polarized laser fluorescence does not give an unambiguous measure of the ground state multipolar moments and in succeeding papers we discuss methods which do yield these quantities without ambiguity.

On the measurement of molecular anisotropies using laser techniques. III. Detection of the higher multipoles

In this paper we discuss the problem of measuring the higher moments (K≥2) of a generalized anisotropic distribution of molecular rotors. Two photon absorption techniques may be used to obtain these quantities and the appropriate expressions for linear and circular dichroism are derived.

Picosecond polarization spectroscopy as a probe of intramolecular dynamics: rovibronic relaxation in the S1 state of trans-stilbene

The first application of picosecond polarization spectroscopy to probe gas phase intramolecular relaxation under collision free conditions is reported. This technique is a sensitive probe of molecular rotation and when applied to trans-stilbene vapor at 200°C an exponential decay in the anisotropy with a lifetime of 48 ± 8 ps is observed. The results are discussed in the context of recent work on IVR and rovibrational intramolecular processes.

Strong molecular alignment in anisotropic fluid media

Abstract Picosecond polarised fluorescence studies of rhodamine 6G molecules in a 100 μm free ethylene glycol jet show the production of strong molecular alignment with a degree of ordering between one to two orders of magnitude greater than that attainable for similar systems using conventional fluid alignment techniques. Using a novel variation of the polarised picosecond single-photon counting technique, it has been possible to prepare a range of non-equilibrium molecular orientational distributions in the jet and observe their subsequent evolution. The imposition of order in the flow is seen to have a distinct effect on molecular motion, indicating a strong anisotropy in θ and /gf rotational diffusion dynamics.