Vladimir P. Denisov

Protein hydration dynamics in aqueous solution

Water oxygen-17 and deuteron spin relaxation rates, measured as a function of resonance frequency, have been used to study the dynamics of protein hydration in aqueous solutions of ribonuclease A, lysozyme, myoglobin, trypsin and serum albumin. The relaxation data conform to the picture of protein hydration dynamics, proposed on the basis of previous studies of smaller proteins, where the long-lived water molecules responsible for the relaxation dispersion are identified with a small number of integrat water molecules seen in the crystal structures. These integral water molecules, with residence times in the range 10(-9)-10(-3) s, are either buried in internal cavities, trapped in narrow clefts or coordinated to metal ions. For the water molecules in the traditional hydration layer at the protein surface, the relaxation data suggest an average residence time in the range 10-50 ps, consistent with high-resolution 1H spectroscopy and computer simulations. The relaxation data also reveal some more specific features of protein hydration, relating to hydration of cavities that appear empty by crystallography, entrapment of water between structural domains of large proteins and subnanosecond 180 degrees flips in buried water clusters.

Hydration of denatured and molten globule proteins.

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of α-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.

Proton NMR of (15)N-choline metabolites enhanced by dynamic nuclear polarization.

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from (15)N to methyl- and methylene protons, using the small (2,3)J((1)H,(15)N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T(1)((15)N) approximately 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.

Feasibility of in vivo N-15 MRS detection of hyperpolarized N-15 labeled choline in rats

The increase of total choline in tumors has become an important biomarker in cancer diagnosis. Choline and choline metabolites can be measured in vivo and in vitro using multinuclear MRS. Recent in vivo(13)C MRS studies using labeled substrates enhanced via dynamic nuclear polarization demonstrated the tremendous potential of hyperpolarization for real-time metabolic studies. The present study demonstrates the feasibility of detecting hyperpolarized (15)N labeled choline in vivo in a rat head at 9.4 T. We furthermore report the in vitro (172 +/- 16 s) and in vivo (126 +/- 15 s) longitudinal relaxation times. We conclude that with appropriate infusion protocols it is feasible to detect hyperpolarized (15)N labeled choline in live animals.

[7] – Magnetic Relaxation Dispersion Studiesof Biomolecular Solutions

Although the MRD method has a long record in biomolecular systems, it has undergone a renaissance in the past few years as methodological developments have provided access to new types of information. In particular, MRD studies of quadrupolar nuclei such as 17O and 23Na have yielded valuable insights about the interactions of proteins and oligonucleotides with their solvent environment. The biomolecular MRD literature is still dominated by hydration studies, but the method has also been used to study the interaction of organic cosolvents and inorganic counterions with biomolecules. The MRD method can potentially make important contributions to the understanding of the mechanisms whereby protein conformational stability is affected by nonaqueous solvent components, such as denaturants, stabilizers, and helix promoters. Residence times of water molecules and other low molecular weight species in association with biomolecules can be determined by MRD. Such residence times are of general interest for understanding the kinetics of biomolecule-ligand interactions and, when exchange is gated by the biomolecule, can be used to characterize large-scale conformational fluctuations on nanosecond-millisecond time scales. By monitoring the integrity and specific internal hydration sites as well as the global solvent exposure, the MRD method can also shed light on the structure and dynamics of biomolecules in fluctuating nonnative states. Because it does not rely on high resolution, the MRD method is also applicable to very large biomolecules and complexes and has even been used to investigate protein crystals, gels, and biological tissues. In fact, dynamic studies of solids and liquid crystals were among the earliest applications of the MRD method. In many of its diverse applications, the MRD method provides unique information, complementing that available from high-resolution NMR.

Early changes in the hypothalamic region in prodromal Huntington disease revealed by MRI analysis.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat. Its length can be used to estimate the time of clinical diagnosis, which is defined by overt motor symptoms. Non-motor symptoms begin before motor onset, and involve changes in hypothalamus-regulated functions such as sleep, emotion and metabolism. Therefore we hypothesized that hypothalamic changes occur already prior to the clinical diagnosis. We performed voxel-based morphometry and logistic regression analyses of cross-sectional MR images from 220 HD gene carriers and 75 controls in the Predict-HD study. We show that changes in the hypothalamic region are detectable before clinical diagnosis and that its grey matter contents alone are sufficient to distinguish HD gene carriers from control cases. In conclusion, our study shows, for the first time, that alterations in grey matter contents in the hypothalamic region occur at least a decade before clinical diagnosis in HD using MRI.

A new view of water dynamics in immobilized proteins

The inflection frequency of the deuteron magnetic relaxation dispersion from water in rotationally immobilized protein samples has recently been found to be essentially independent of temperature and protein structure. This remarkable invariance has been interpreted in terms of a universal residence time of 1 microseconds for protein-associated water molecules. We demonstrate here that this interpretation is an artifact of the conventional perturbation theory of spin relaxation, which is not valid for rotationally immobile proteins. Using a newly developed non-perturbative, stochastic theory of spin relaxation, we identify the apparent correlation time of 1 microseconds with the inverse of the nuclear quadrupole frequency, thus explaining its invariance. The observed dispersion profiles are consistent with a broad distribution of residence times, spanning the microseconds range. Furthermore, we argue that the deuteron dispersion is due to buried water molecules rather than to the traditional surface hydration previously invoked, and that the contribution from rapidly exchanging protein hydrogens cannot be neglected. The conclusions of the present work are also relevant to proton relaxation in immobilized protein samples and to magnetic resonance imaging of soft tissue.

Magnetic Resonance Spectroscopic Methods for the Assessment of Metabolic Functions in the Diseased Brain

Magnetic resonance spectroscopy (MRS) is a non-invasive technique that can be used to detect and quantify multiple metabolites. This chapter will review some of the applications of MRS to the study of brain functions. Typically, (1)H-MRS can detect metabolites reflecting neuronal density and integrity, markers of energy metabolism or inflammation, as well as neurotransmitters. The complexity of the proton spectrum has however led to the development of other nuclei-based methods, such as (31)P- and (13)C-MRS, which offer a broader chemical shift range and therefore can provide more detailed information at the level of single metabolites. The versatility of MRS allows for a wide range of clinical applications, of which neurodegeneration is an interesting target for spectroscopy-based studies. In particular, MRS can identify patterns of altered brain chemistry in Alzheimers patients and can help establish differential diagnosis in Alzheimers and Parkinsons diseases. Using MRS to follow less abundant neurotransmitters is currently out of reach and will most likely depend on the development of methods such as hyperpolarization that can increase the sensitivity of detection. In particular, dynamic nuclear polarization has opened up a new and exciting area of medical research, with developments that could greatly impact on the real-time monitoring of in vivo metabolic processes in the brain.

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimers and Parkinsons diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post‐mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used 15N‐choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before 1H‐NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT‐mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real‐time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non‐invasive manner using NMR spectroscopic techniques.