Scholarly Work - Chemistry & Biochemistry
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8714
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Item Two-photon absorption spectra of fluorescent isomorphic DNA base analogs(2018-01) Mikhaylov, Alexander E.; Reguardati, Sophie de; Pahapill, Juri; Callis, Patrik R.; Kohler, Bern; Rebane, AleksanderFluorescent DNA base analogs and intrinsic fluorophores are gaining importance for multiphoton microscopy and imaging, however, their quantitative nonlinear excitation properties have been poorly documented. Here we present the two-photon absorption (2PA) spectra of 2-aminopurine (2AP), 7-methyl guanosine (7MG), isoxanthopterin (IXP), 6-methyl isoxanthopterin (6MI), as well as L-tryptophan (L-trp) and 3-methylindole (3MI) in aqueous solution and some organic solvents measured in the wavelength range 550 - 810 nm using femtosecond two-photon excited fluorescence (2PEF) and nonlinear transmission (NLT) methods. The peak 2PA cross section values range from 0.1 GM (1 GM = 10-50 cm4 s photon-1) for 2AP to 2.0 GM for IXP and 7MG. Assuming typical excitation conditions for a scanning 2PEF microscope, we estimate a maximum image frame rate of ~175 frames per second (FPS).Item Picosecond Fluorescence Dynamics of Tryptophan and 5-Fluorotryptophan in Monellin: Slow Water-Protein Relaxation Unmasked(2015-03) Xu, Jianhua; Chen, Binbin; Callis, Patrik R.; Muino, Pedro L.; Rozenboom, Henriette; Broos, Jaap; Toptygin, Dmitri; Brand, Ludwig; Knutson, Jay R.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 the sweet-tasting protein monellin which has uniquely been the subject of ultrafast studies in two different laboratories (Peon, J.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10964; Xu, J.; et al. J. Am. Chem. Soc. 2006, 128, 1214) that led to disparate interpretations of a 20 ps transient. They differed because of the pseudo-TDFSS present. The current study exploiting special properties of 5FTrp strongly supports the conclusion that both lifetime heterogeneity-based TDFSS and environment relaxation-based TDFSS are present in monellin and 5FTrp-monellin. The original experiments on monellin were most likely dominated by pseudo-TDFSS, whereas, in the present investigation of 5FTrp-monellin, the TDFSS is dominated by relaxation and any residual pseudo-TDFSS is overwhelmed and/or slowed to irrelevance.Item Simulating electrostatic effects on electronic transitions in proteins(2014-06) Callis, Patrik R.Biopolymer fluorescence in biology and biochemistry is increasingly used for characterising equilibrium, dynamics and imaging. This is typically done by monitoring wavelength and intensity changes without necessarily knowing what causes such changes in detail. Simulations have been at the core of the considerable recent progress in improving the microscopic understanding of wavelength and quenching of fluorescence intensity in biopolymers. This review focuses on one of the most used intrinsic probes for protein behaviour, tryptophan (Trp), which is arguably now one of the best understood probes of internal structure and dynamics for proteins – despite its reputation to the contrary. In this review, we highlight selected classical molecular dynamics in combination with quantum mechanics simulations from our group and others during the past 20 years that support this view. The work includes simulations of time-dependent wavelength shifts in solvents and proteins, fluorescence-quenching rates, dielectric compensation by water, heterogeneity of quenching rates and applications to protein folding.