Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry;it can provide unique insights into the structure and assembly of macromolecular complexes. This year, we pursued protein foldingand ultrafast protein solvation. We continued and expanded our femtosecond upconversion studies of Trp in proteins and peptides to quantify early """"""""quasistatic self-quenching"""""""" processes that confound studies of solvation dynamics. We previously published evidence that extremely rapid (10-100ps) decays are important in several proteins (crystallins, thioredoxin, etc.), as they detect previously silent conformers engaged in ultrafast charge transfer. Our earlier study of protein *solvation* on the 330fs-200ps time scale, using proteins such as Monellin, found QSSQ that others attributed to a class of unique water molecules that desorb from protein in 20ps. Local quenching is the dominant mechanism in all but a few cases we have studied. The QSSQ was found even in simple dipeptides ,suggesting a general process underlies all protein QSSQ. We explored the slow relaxation (50ps) of water in the protein GB1 as a function of pH and temperature, helping to understand why this one small protein lacks the QSSQ that usually masks relaxation. At the same time, we have determined the relaxation is not coupled to local solvent access of Trp (measured during pH titration with soluble quenchers and also seen in ns lifetimes). This also showed that the charge environment changes accompanying titration did not change the local mobility of water. We also began studying Trp analogs that , almost immune to QSSQ, respond to relaxing water matrices without masking by local electron transfer kinetics. The titration study is in minor revision stages at JACS;the overall subject of upconversion (sub-ps) studies of Trp was published by us in a book chapter. We contined collaborative studies with LCE into the status of a primary fuel of heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and in collaboration with Light Microscopy Core and LCE, we are continually refining 'Decay-Associated Images'software to more rapidly extract profiles of NADH binding within isolated cardiac myocytes. This year we added NADH studies in melanoma models. We continue to develop coupled lifetime and translational diffusion capabilities in time-resolved FCS for this and other projects.

Project Start
Project End
Budget Start
Budget End
Support Year
30
Fiscal Year
2013
Total Cost
$325,821
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Rosales, Tilman; Sackett, Dan L; Xu, Jianhua et al. (2015) STAQ: A route toward low power, multicolor nanoscopy. Microsc Res Tech 78:343-55
Xu, Jianhua; Chen, Binbin; Callis, Patrik et al. (2015) Picosecond fluorescence dynamics of tryptophan and 5-fluorotryptophan in monellin: slow water-protein relaxation unmasked. J Phys Chem B 119:4230-9
Biesso, Arianna; Xu, Jianhua; Knutson, Jay R (2014) Upconversion spectrophotofluorometry. Methods Mol Biol 1076:303-19
Combs, C A; Smirnov, A; Glancy, B et al. (2014) Compact non-contact total emission detection for in vivo multiphoton excitation microscopy. J Microsc 253:83-92
Biesso, Arianna; Xu, Jianhua; Muino, Pedro L et al. (2014) Charge invariant protein-water relaxation in GB1 via ultrafast tryptophan fluorescence. J Am Chem Soc 136:2739-47
Dasgeb, Bahar; Smirnov, Aleksandr V; Ardeshirpour, Yasaman et al. (2014) Multiscale BerEp4 molecular imaging of microtumor phantoms: toward Theranostics for basal cell Carcinoma. Mol Imaging 13:
Rosales, Tilman; Nie, Zuqin; Kapoor, Varun et al. (2013) Partition of Myc into Immobile vs. Mobile Complexes within Nuclei. Sci Rep 3:1953
Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal et al. (2012) In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers. PLoS One 7:e31881
Ben-Aissa, Khadija; Patino-Lopez, Genaro; Belkina, Natalya V et al. (2012) Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker. J Biol Chem 287:16311-23
Combs, C A; Smirnov, A; Chess, D et al. (2011) Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED). J Microsc 241:153-61

Showing the most recent 10 out of 17 publications