Jianhua Xu

Femtosecond Fluorescence Spectra of Tryptophan in Human γ-Crystallin Mutants: Site-Dependent Ultrafast Quenching

The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.

Quasi-Static Self-Quenching of Trp-X and X-Trp Dipeptides in Water: Ultrafast Fluorescence Decay

Time-resolved fluorescence decay profiles of N-acetyl-l-tryptophanamide (NATA) and tryptophan (Trp) dipeptides of the form Trp-X and X-Trp, where X is another aminoacyl residue, have been investigated using an ultraviolet upconversion spectrophoto fluorometer with time resolution better than 350 fs, together with a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. We analyzed the set of fluorescence decay profiles at multiple wavelengths using the global analysis technique. Nanosecond fluorescence transients for Trp dipeptides all show multiexponential decay, while NATA exhibits a monoexponential decay near 3 ns independent of pH. In the first 100 ps, a time constant for the water bulk relaxation around Trp, NATA and Trp dipeptides are seen near 1-2 ps, with an associated preexponential amplitude that is positive or negative, depending on emission wavelength, as expected for a population conserving spectral shift. The initial brightness (sub-picosecond) we measure for all these dipeptides is less than that of NATA, implying even faster (<200 fs) intramolecular (quasi-) static quenching occurs within them. A new, third, ultrafast decay, bearing an exponential time constant of 20-30 ps with positive amplitude, has been found in many of these dipeptides. We believe it verifies our previous predictions of dipeptide QSSQ (quasi-static self-quenching)-the loss of quantum yield to sub-100-ps decay process (Chen, R. F.; et al. Biochemistry 1991, 30, 5184). Most important, this term is found in proteins as well (Xu, J.; et al. J. Am. Chem. Soc. 2006, 128, 1214; Biophys. J. 2008, 94, 546; 2009, 96, 46a), suggesting an ultrafast quenching mechanism must be common to both.

Quantitative detection of the ligand-dependent interaction between the androgen receptor and the co-activator, Tif2, in live cells using two color, two photon fluorescence cross-correlation spectroscopy

Two-photon, two-color fluorescence cross-correlation spectroscopy (TPTCFCCS) was used to directly detect ligand-dependent interaction between an eCFP-fusion of the androgen receptor (eCFP-AR) and an eYFP fusion of the nuclear receptor co-activator, Tif2 (eYFP-Tif2) in live cells. As expected, these two proteins were co-localized in the nucleus in the presence of ligand. Analysis of the cross-correlation amplitude revealed that AR was on average 81% bound to Tif2 in the presence of agonist, whereas the fractional complex formation decreased to 56% in the presence of antagonist. Residual AR–Tif2 interaction in presence of antagonist is likely mediated by its ligand-independent activation function. These studies demonstrate that using TPTCFCCS it is possible to quantify ligand-dependent interaction of nuclear receptors with co-regulator partners in live cells, making possible a vast array of structure-function studies for these important transcriptional regulators.

Ultrafast fluorescence spectroscopy via upconversion applications to biophysics.

This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed.

Molecular Dynamics Simulations of Perylene and Tetracene Librations: Comparison With Femtosecond Upconversion Data

In a prior manuscript by Xu et al. [Xu, J.; Shen, X.; Knutson, J. R. J. Phys. Chem. A 2003, 107, 8383], time-resolved fluorescence emission anisotropy measurements were performed on perylene and tetracene in hexadecane using an upconversion technique with approximately 100 fs resolution. The anisotropy transients contained previously unseen decay terms of approximately 300 fs. In perylene, their amplitude corresponded to the r(o) defect that has gathered interest over decades. We ascribed this term to a predominantly in-plane libration. In this manuscript, we present molecular dynamics simulations for the motions of perylene and tetracene using the CHARMm molecular dynamics program (version c29b2). Both rotational correlation functions contain subpicosecond decay terms that resemble experimental anisotropy decays. It was suggested that the r(o) defect might arise from excited-state distortions of perylene, so we conducted quantum mechanical calculations to show that such distortion does not significantly displace the oscillators. We compare the case of perylene, with a strongly allowed singlet emission transition, to that of the weakly allowed tetracene transition. In perylene, motion alone can explain subpicosecond anisotropy decay, while tetracene decay also contains vibrational coupling terms, as previously reported by Sarkar et al. [Sarkar, N.; Takeuchi, S.; Tahara, T. J. Phys. Chem. A 1999, 103, 4808].

Charge invariant protein-water relaxation in GB1 via ultrafast tryptophan fluorescence.

The protein–water interface is a critical determinant of protein structure and function, yet the precise nature of dynamics in this complex system remains elusive. Tryptophan fluorescence has become the probe of choice for such dynamics on the picosecond time scale (especially via fluorescence “upconversion”). In the absence of ultrafast (“quasi-static”) quenching from nearby groups, the TDFSS (time-dependent fluorescence Stokes shift) for exposed Trp directly reports on dipolar relaxation near the interface (both water and polypeptide). The small protein GB1 contains a single Trp (W43) of this type, and its structure is refractory to pH above 3. Thus, it can be used to examine the dependence of dipolar relaxation upon charge reconfiguration with titration. Somewhat surprisingly, the dipolar dynamics in the 100 fs to 100 ps range were unchanged with pH, although nanosecond yield, rates, and access all changed. These results were rationalized with the help of molecular dynamics (including QM-MM) simulations that reveal a balancing, sometimes even countervailing influence of protein and water dipoles. Interestingly, these simulations also showed the dominant influence of water molecules which are associated with the protein interface for up to 30 ps yet free to rotate at approximately “bulk” water rates.

Picosecond Fluorescence Dynamics of Tryptophan and 5-Fluorotryptophan in Monellin: Slow Water-Protein Relaxation Unmasked

Time dependent fluorescence Stokes (emission wavelength) shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many fluorescence probes, both the efficiency and the wavelength of Trp fluorescence in proteins are highly sensitive to microenvironment, and Stokes shifts can be dominated by the well-known heterogeneous nature of protein structure, leading to what we call pseudo-TDFSS: shifts that arise from differential decay rates of subpopulations. Here we emphasize a novel, general method that obviates pseudo-TDFSS by replacing Trp by 5-fluorotryptophan (5Ftrp), a fluorescent analogue with higher ionization potential and greatly suppressed electron-transfer quenching. 5FTrp slows and suppresses pseudo-TDFSS, thereby providing a clearer view of genuine relaxation caused by solvent and protein response. This procedure is applied to ...

STAQ: A route toward low power, Multicolor nanoscopy

Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real‐time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (∼50 nm) imaging. Moreover, the TAQ partner excited by the “donut” beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real‐time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopists palette. Microsc. Res. Tech. 78:343–355, 2015. Published 2015. This article is a U.S. Government work and is in the public domain in the USA

The Impact of Laser Evolution on Modern Fluorescence Spectroscopy

The judicious use of traditional spectroscopy light sources throughout the postwar era led to the foundations of fluorescence spectroscopy, both theoretically and experimentally. Those principles provided many tools for understanding the structure and dynamics of macromolecules, cells, and even tissues. In the last four decades those tools have been supplemented and sometimes extended by the availability of novel light sources, advanced electronics, and burgeoning computing power. This chapter will chronicle the former – the impact of four decades of laser evolution upon biological fluorescence spectroscopy and microscopy. It is necessarily focused on only the systems that were most popular and influential (many other sources were of great value) and (for space concerns) it also summarizes only a few of the many linked technological advances.

Is 10-100ps Spectral Relaxation of Trp An Indicator of Local Disorder in Proteins?

We have studied the time-resolved fluorescence of the Trp43 residue of the globular protein GB1 upon acid induced equilibrium unfolding.NMR structural experiments have shown this protein is actually very acid stable above pH3.Nanosecond time-resolved TCSPC data clearly suggest that, in the tryptophan environment, partial unfolding appears at surprisingly high pH values. In fact, GB1 exhibits signs of local lifetime changes for pH values as high as 6.9.Further, femtosecond ultraviolet upconversion data reveal a ∼30 ps component with a negative preexponential amplitude in the longer wavelength portion of the emission spectrum. Such a rise time on the red side of emission is a signature of generalized relaxation (solvent and/or protein) around the excited dipole of Trp. A similar term with a ∼2ps exponential is always found for proteins in aqueous solution, representing bulk water motion. Slower terms (10-100ps) have previously been assigned to unusual water environments, protein dipolar relaxation, or the coupling between them.Most intriguing is the fact that, for somewhat lower pH values where GB1 is locally (but not globally) unfolded to a larger degree, the amplitude of the observed 30ps term becomes larger (more negative).Femtosecond Trp emission spectroscopy may thus provide new snapshots of proteins that are “fully folded” over longer time averaging but still have transiently unstructured regions.