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.

A distribution-based method to resolve single-molecule Förster resonance energy transfer observations.

We introduce a new approach to analyze single-molecule Förster resonance energy transfer (FRET) data. The method recognizes that FRET efficiencies assumed by traditional ensemble methods are unobservable for single molecules. We propose instead a method to predict distributions of FRET parameters obtained directly from the data. Distributions of FRET rates, given the data, are precisely defined using Bayesian methods and increase the information derived from the data. Benchmark comparisons find that the response time of the new method outperforms traditional methods of averaging. Our approach makes no assumption about the number or distribution of underlying FRET states. The new method also yields information about joint parameter distributions going beyond the standard framework of FRET analysis. For example, the running distribution of FRET means contains more information than any conceivable single measure of FRET efficiency. The method is tested against simulated data and then applied to a pilot-study sample of calmodulin molecules immobilized in lipid vesicles, revealing evidence for multiple dynamical states.

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.

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.

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.

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

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta 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 delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), 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 PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), as well as from exogenous CaM bound to the PhK complex as delta'. The distinction between delta and delta' is that the latter binds only in the presence of Ca(2+), but stoichiometrically and at a different location in the complex than delta.

Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy.

This work evaluates a femtosecond fiber laser for use in two-photon fluorescence fluctuation spectroscopy. Fiber lasers present an attractive alternative to Ti:Sapphire systems because of their compact size and portability. Autocorrelation of the second harmonic generation signal from the laser demonstrates that its stability is sufficient for two-photon fluorescence correlation spectroscopy. Fluorescence correlation spectroscopy autocorrelation traces were well fit by a Gaussian-Lorentzian squared model with a beam waist near the diffraction limit for the 810 nm wavelength. A photon counting histogram collected with this system also fit nicely to a single-species model, further demonstrating the quality of the focal shape. The authors conclude that the output from the femtosecond fiber laser is sufficiently stable and has a high enough quality beam shape for fluctuation fluorescence methods, and thus represents an effective, compact, readily portable two-photon excitation source.

Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy.

We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially.