Two-photon excitation microscopy of tryptophan-containing proteins

Abstract

We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

Stopped-flow Fluorescence Studies of Inhibitor Binding to Tyrosinase from Streptomyces antibioticus

“…The positions of the emission maxima (343 Ϯ 2 nm for all species) are the highest reported for a type 3 copper protein and indicate that a significant fraction of the Trp residues is exposed to the solvent. As was found earlier for several hemocyanins (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39) and Neurospora crassa tyrosinase (40), it appears that the Ty fluorescence emission intensities are markedly sensitive to the oxidation state of the dinuclear copper center; the emission quantum yields of Ty red and Ty oxy differ by more than a factor of 2.…”

“…Several Hcs (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39) and one Ty (40) have been the subject of such investigations. Yet Hc or Ty protein fluorescence has never been used to study inhibitor binding.…”

Photoreactivity of Amino Acids: Tryptophan-induced Photochemical Events via Reactive Oxygen Species Generation

“…Recently, there have been reports indicating that some Trp-containing proteins showed photochemical reactions, such as photodegradation, dimerization, and photoionization. [6][7][8] Although the photostability of Trpcontaining peptides/proteins has been investigated, the pathway for photochemical reactions induced by Trp has not yet been fully elucidated.…”

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

“…Here, we aim to systematically characterize not only the mechanism and dynamics involved in product release of HRP but also the complex conformational dynamics involved in each step of a catalytic cycle by directly manipulating the reaction coordinates. We further extended our study and applied our newly developed SM approach of actively manipulating the reaction coordinates using combined SM time-resolved photon time-stamping spectroscopy and magnetic tweezers to understand the effect of conformational perturbation on the conformational dynamics and mechanism of enzymatic product release.The biological function of an enzyme is presumably followed in real time by using mainly three kinds of fluorescent reporter molecules: (i) intrinsic fluorescent residues (e.g., tryptophan or tyrosine), (ii) site-specific labeling, and (iii) fluorogenic substrate (8,11,21). Application of fluorogenic substrate (nonfluorescent) is a direct approach to follow enzymatic reaction turnovers as it gets converted into a fluorescent product molecule by the enzyme.…”

“…The biological function of an enzyme is presumably followed in real time by using mainly three kinds of fluorescent reporter molecules: (i) intrinsic fluorescent residues (e.g., tryptophan or tyrosine), (ii) site-specific labeling, and (iii) fluorogenic substrate (8,11,21). Application of fluorogenic substrate (nonfluorescent) is a direct approach to follow enzymatic reaction turnovers as it gets converted into a fluorescent product molecule by the enzyme.…”