Eugene Milshteyn

Characterization of the disordered‐to‐α‐helical transition of IA3 by SDSL‐EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy coupled with site‐directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA3 is used as a model system demonstrating SDSL‐EPR characterization of conformational changes in small IDP systems. IA3 has 68 amino acids, is unstructured in solution, and becomes α‐helical upon addition of the secondary structural stabilizer 2,2,2‐trifluoroethanol (TFE). Two single cysteine substitutions, one in the N‐terminus (S14C) and one in the C‐terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h(+1)/h(0), the local tumbling volume (VL), and the percent change of the h(−1) intensity were utilized to quantitatively monitor TFE‐induced conformational changes. The values of h(+1)/h(0) as a function of TFE titration varied in a sigmoidal manner and were fit to a two‐state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA3. The other parameters provide site‐specific information and show that S14C‐SL undergoes a conformational change resulting in more restricted motion than N58C‐SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α‐helical propensity of the N‐terminal segment of IA3. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured‐to‐helical conformations in IDPs by SDSL.

Handheld electromagnet carrier for transfer of hyperpolarized carbon-13 samples.

Hyperpolarization of carbon‐13 (13C) nuclei by dissolution dynamic nuclear polarization increases signal‐to‐noise ratio (SNR) by >10,000‐fold for metabolic imaging, but care must be taken when transferring hyperpolarized (HP) samples from polarizer to MR scanner. Some 13C substrates relax rapidly in low ambient magnetic fields. A handheld electromagnet carrier was designed and constructed to preserve polarization by maintaining a sufficient field during sample transfer.

Monitoring acute metabolic changes in the liver and kidneys induced by fructose and glucose using hyperpolarized [2-13C]dihydroxyacetone

To investigate acute changes in glucose metabolism in liver and kidneys in vivo after a bolus injection of either fructose or glucose, using hyperpolarized [2‐13C]dihydroxyacetone.

Spectrally selective three-dimensional dynamic balanced steady-state free precession for hyperpolarized C-13 metabolic imaging with spectrally selective radiofrequency pulses

Balanced steady‐state free precession (bSSFP) sequences can provide superior signal‐to‐noise ratio efficiency for hyperpolarized (HP) carbon‐13 (13C) magnetic resonance imaging by efficiently utilizing the nonrecoverable magnetization, but managing their spectral response is challenging in the context of metabolic imaging. A new spectrally selective bSSFP sequence was developed for fast imaging of multiple HP 13C metabolites with high spatiotemporal resolution.

Continuous wave W- and D-band EPR spectroscopy offer "sweet-spots" for characterizing conformational changes and dynamics in intrinsically disordered proteins.

Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for characterizing conformational sampling and dynamics in biological macromolecules. Here we demonstrate that nitroxide spectra collected at frequencies higher than X-band (∼9.5 GHz) have sensitivity to the timescale of motion sampled by highly dynamic intrinsically disordered proteins (IDPs). The 68 amino acid protein IA3, was spin-labeled at two distinct sites and a comparison of X-band, Q-band (35 GHz) and W-band (95 GHz) spectra are shown for this protein as it undergoes the helical transition chemically induced by tri-fluoroethanol. Experimental spectra at W-band showed pronounced line shape dispersion corresponding to a change in correlation time from ∼0.3 ns (unstructured) to ∼0.6 ns (α-helical) as indicated by comparison with simulations. Experimental and simulated spectra at X- and Q-bands showed minimal dispersion over this range, illustrating the utility of SDSL EPR at higher frequencies for characterizing structural transitions and dynamics in IDPs.

Mis‐estimation and bias of hyperpolarized apparent diffusion coefficient measurements due to slice profile effects

The purpose of this work was to explore the impact of slice profile effects on apparent diffusion coefficient (ADC) mapping of hyperpolarized (HP) substrates.

Development of high resolution 3D hyperpolarized carbon-13 MR molecular imaging techniques

The goal of this project was to develop and apply techniques for T2 mapping and 3D high resolution (1.5mm isotropic; 0.003cm3) 13C imaging of hyperpolarized (HP) probes [1-13C]lactate, [1-13C]pyruvate, [2-13C]pyruvate, and [13C,15N2]urea in vivo. A specialized 2D bSSFP sequence was implemented on a clinical 3T scanner and used to obtain the first high resolution T2 maps of these different hyperpolarized compounds in both rats and tumor-bearing mice. These maps were first used to optimize timings for highest SNR for single time-point 3D bSSFP acquisitions with a 1.5mm isotropic spatial resolution of normal rats. This 3D acquisition approach was extended to serial dynamic imaging with 2-fold compressed sensing acceleration without changing spatial resolution. The T2 mapping experiments yielded measurements of T2 values of >1s for all compounds within rat kidneys/vasculature and TRAMP tumors, except for [2-13C]pyruvate which was ~730ms and ~320ms, respectively. The high resolution 3D imaging enabled visualization the biodistribution of [1-13C]lactate, [1-13C]pyruvate, and [2-13C]pyruvate within different kidney compartments as well as in the vasculature. While the mouse anatomy is smaller, the resolution was also sufficient to image the distribution of all compounds within kidney, vasculature, and tumor. The development of the specialized 3D sequence with compressed sensing provided improved structural and functional assessments at a high (0.003cm3) spatial and 2s temporal resolution in vivo utilizing HP 13C substrates by exploiting their long T2 values. This 1.5mm isotropic resolution is comparable to 1H imaging and application of this approach could be extended to future studies of uptake, metabolism, and perfusion in cancer and other disease models and may ultimately be of value for clinical imaging.

Non-invasive detection of divergent metabolic signals in insulin deficiency vs. insulin resistance in vivo

The type 2 diabetic phenotype results from mixed effects of insulin deficiency and insulin resistance, but the relative contributions of these two distinct factors remain poorly characterized, as do the respective roles of the gluconeogenic organs. The purpose of this study was to investigate localized in vivo metabolic changes in liver and kidneys of contrasting models of diabetes mellitus (DM): streptozotocin (STZ)-treated wild-type Zucker rats (T1DM) and Zucker diabetic fatty (ZDF) rats (T2DM). Intermediary metabolism was probed using hyperpolarized (HP) [1-13C]pyruvate MRI of the liver and kidneys. These data were correlated with gene expression data for key mediators, assessed using rtPCR. Increased HP [1-13C]lactate was detected in both models, in association with elevated gluconeogenesis as reflected by increased expression of phosphoenolpyruvate carboxykinase. In contrast, HP [1-13C]alanine diverged between the two models, increasing in ZDF rats, while decreasing in the STZ-treated rats. The differences in liver alanine paralleled differences in key lipogenic mediators. Thus, HP [1-13C]alanine is a marker that can identify phenotypic differences in kidneys and liver of rats with T1DM vs. T2DM, non-invasively in vivo. This approach could provide a powerful diagnostic tool for characterizing tissue metabolic defects and responses to treatment in diabetic patients with ambiguous systemic manifestations.

A regional bolus tracking and real-time B1 calibration method for hyperpolarized 13C MRI

Acquisition timing and B1 calibration are two key factors that affect the quality and accuracy of hyperpolarized 13C MRI. The goal of this project was to develop a new approach using regional bolus tracking to trigger Bloch‐Siegert B1 mapping and real‐time B1 calibration based on regional B1 measurements, followed by dynamic imaging of hyperpolarized 13C metabolites in vivo.

Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA 3

IA 3 is an intrinsically disordered protein (IDP) that becomes helical when bound to yeast proteinase A (YPRA) or in the presence of the secondary stabilizer 2,2,2-trifluoroethanol (TFE). Here, site-directed spin-labeling (SDSL) continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy and circular dichroism (CD) are used to characterize the TFE-induced helical conformation of IA 3 for a series of spin-labeled cysteine scanning constructs and varied amino acid substitutions. Results demonstrate that the N-terminal concave helical surface of IA 3 , which is the buried interface when bound to YPRA, can be destabilized by the spin-label or bulky amino acid substitutions. In contrast, the helical tendency of IA 3 is enhanced when spin-labels are incorporated into the convex, i.e., solvent exposed, surface of IA 3 . No impact of the spin-label within the C-terminal region was observed. This work further demonstrates the utility and sensitivity of SDSL CW-EPR for studies of IDPs. In general, care must be taken to ensure the spin-label does not interfere with native helical tendencies and these studies provide us with knowledge of where to incorporate spin-labels for future SDSL investigations of IA 3 .