E. Shane Price

Two-photon microscopy with wavelength switchable fiber laser excitation

Two-photon scanning fluorescence microscopy has become a powerful tool for imaging living cells and tissues. Most applications of two-photon microscopy employ a Ti:sapphire laser excitation source, which is not readily portable or rapidly tunable. This work explores the use of two-photon fiber laser excitation (TP-FLEX) as an excitation source for scanning two-photon microscopy. We have further demonstrated the use of a photonic crystal fiber (PCF) for facile tuning of the excitation wavelength over the range from 810 nm to 1100 nm. We generated two-photon fluorescence images at excitation wavelengths from 850 nm to 1100 nm detected on a scanning-stage microscope. By PCF wavelength tuning the dye BODIPY fl was selectively excited at 1000 nm whereas MitoTracker red was excited preferentially at 1100 nm. We discuss the potential for fiber laser sources coupled with PCF wavelength tuning as an attractive tunable excitation source for two-photon scanning fluorescence microscopy.

Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability

Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic EPDs. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed.

Detecting intramolecular dynamics and multiple Förster resonance energy transfer states by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for the detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (green-red and red-green). We applied the method to detect intramolecular dynamics in the Ca(2+) signaling protein calmodulin. Dynamics were detected on the 100 mus time scale in Ca(2+)-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro(15)-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto- and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca(2+)-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.

FRET-FCS detection of intralobe dynamics in calmodulin.

Fluorescence correlation spectroscopy (FCS) can be coupled with Förster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-FCS to detect fluctuations within the N-terminal and C-terminal domains of the Ca(2+)-signaling protein calmodulin. Intramolecular fluctuations were resolved by global fitting of the two fluorescence autocorrelation functions (green-green and red-red) together with the two cross-correlation functions (green-red and red-green). To match the Förster radius for FRET to the dimensions of the N-terminal and C-terminal domains, a near-infrared acceptor fluorophore (Atto 740) was coupled with a green-emitting donor (Alexa Fluor 488). Fluctuations were detected in both domains on the time scale of 30 to 40 μs. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca(2+) binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca(2+)) was nearly abolished at a high Ca(2+) concentration. For the C-terminal domain, the dynamic amplitude changed little with Ca(2+) concentration. The Ca(2+) dependence of dynamics for the N-terminal domain suggests that the fluctuations detected by FCS in the N-terminal domain are coupled to the opening and closing of the EF-hand Ca(2+)-binding loops.

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral δ subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca2+ sensitivity to the enzyme, but bind independently of Ca2+. In addition to binding Ca2+, an obligatory activator of PhKs phosphoryltransferase activity, the δ subunits transmit allosteric signals to PhKs remaining α, β, and γ subunits in activating the enzyme. Under mild conditions about 10% of the δ subunits can be exchanged for exogenous CaM. In this study, a CaM double‐mutant derivatized with a fluorescent donor–acceptor pair (CaM‐DA) was exchanged for δ to assess the conformational substates of PhKδ by single molecule fluorescence resonance energy transfer (FRET) ±Ca2+. The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca2+, as observed by alterations of the compact, mid‐length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid‐length and less common compact conformations of PhKδ became less abundant in the presence of Ca2+, with the δ subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaMs Ca2+‐dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKδ subunits were distinct from those of CaM‐DA free in solution, ±Ca2+, as well as from exogenous CaM bound to the PhK complex as δ′. The distinction between δ and δ′ is that the latter binds only in the presence of Ca2+, but stoichiometrically and at a different location in the complex than δ.

Switching of 800 nm femtosecond laser pulses using a compact PMN-PT modulator

A voltage-controlled birefringent cell based on ceramic PMN-PT material is used to enable fast intensity modulation of femtosecond laser pulses in the 800 nm wavelength window. The birefringent cell based on a PMN-PT compound has comparatively high electro-optic response, allowing for a short interaction length of 3 mm and thus very small size, low attenuation of 0.16 dB, and negligible broadening for 100 fs optical pulses. As an application example, agile wavelength tuning of optical pulses is demonstrated using the soliton self-frequency shift in a photonic crystal fiber. By dynamically controlling the optical power into the fiber, this system switches the wavelength of 100 fs pulses from 900 nm to beyond 1120 nm with less than 5 micros time. In addition, a feedback system stabilizes the wavelength drift against external conditions resulting in high wavelength stability.

Fluorescence Probes of Protein Dynamics and Conformations in Freely Diffusing Molecules

The results described here demonstrate that single-molecule fluorescence spectroscopy of molecules freely diffusing in solution can yield unique information about both the dynamics and conformations of proteins. We used single-molecule FRET to detect the dynamics in CaM on the timescales of 100’s of microseconds and a few milliseconds by cross-correlation analysis of the donor and acceptor signals. In addition, analysis of the distribution of donor-acceptor distances by single-molecule FRET demonstrates the presence of three distinct conformational substates of CaM in solution. Together these results paint a picture of a protein that is dynamic and flexible, accessing a range of distinct conformation substates in solution.

Investigations of Calmodulin Conformations Resolved by Single Molecule Microscopy

Measurements of the distance between two dye molecules covalently linked to the calcium signaling protein calmodulin (CaM) have been previously performed by our group to investigate the conformations of CaM in solution. It was shown that calmodulin exists in a wide range of distinct conformations whose amplitudes depend upon free calcium concentrations (1). Currently, we are investigating affects that the choice of dye pair or labeling site has on measured conformations. This is done using an alternating laser excitation (ALEX) single molecule microscope system that has been custom built in our laboratory. Time correlated single photon counting in bulk samples is used to determine the time resolved anisotropy of the dye pair and the orientational mobility of each dye. Analysis of burst measurements using interphoton time burst selection criteria and the probability distribution analysis reveal a wide range of CaM conformations. Conformational analysis is performed using both discrete states and the maximum entropy method. The maximum entropy method reveals the most probable underlying conformational distribution that fits our data. Finally, we are investigating fluorescence fluctuations within CaM conformations using conformationally sorted fluorescence correlation spectroscopy.1. Slaughter et al., J. Phys. Chem. B, 2004, 108, 10388-10397