New NMR methods for the characterization of bound waters in macromolecules (original) (raw)
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Synchrotron X-ray footprinting as a method to visualize water in proteins
Journal of synchrotron radiation, 2016
The vast majority of biomolecular processes are controlled or facilitated by water interactions. In enzymes, regulatory proteins, membrane-bound receptors and ion-channels, water bound to functionally important residues creates hydrogen-bonding networks that underlie the mechanism of action of the macromolecule. High-resolution X-ray structures are often difficult to obtain with many of these classes of proteins because sample conditions, such as the necessity of detergents, often impede crystallization. Other biophysical techniques such as neutron scattering, nuclear magnetic resonance and Fourier transform infrared spectroscopy are useful for studying internal water, though each has its own advantages and drawbacks, and often a hybrid approach is required to address important biological problems associated with protein-water interactions. One major area requiring more investigation is the study of bound water molecules which reside in cavities and channels and which are often invo...
2008
Water plays a critical role in the structure and function of proteins, although the experimental properties of water around protein structures are not well understood. The water can be classified by the separation from the protein surface into bulk water and hydration water. Hydration water interacts closely with the protein and contributes to protein folding, stability and dynamics, as well as interacting with the bulk water. Water potential functions are often parameterized to fit bulk water properties because of the limited experimental data for hydration water. Therefore, the structural and energetic properties of the hydration water were assessed for 105 atomic resolution (≤1.0 Å) protein crystal structures with a high level of hydration water by calculating the experimental water-protein radial distribution function or surface distribution function (SDF) and water radial distribution function (RDF). Two maxima are observed in SDF: the first maximum at a radius of 2.75 Å reflects first shell and hydrogen bond interactions between protein and water, the second maximum at 3.65 Å reflects second shell and van der Waals interactions between water and non-polar atoms of protein forming clathrate-hydrate-like structures. Thus, the two shells do not overlap. The RDF showed the features of liquid water rather than solid ice. The first and second maxima of RDF at 2.75 and 4.5 Å, respectively, are the same as for bulk water, but the peaks are sharper indicating hydration water is more stable than bulk water. Both distribution functions are inversely correlated with the distribution of B factors (atomic thermal factors) for the waters, suggesting that the maxima reflect stable positions. Therefore, the average water structure near the protein surface has experimentally observable differences from bulk water. This analysis will help improve the accuracy for models of water on the protein surface by providing rigorous data for the effects of the apparent chemical potential of the water near a protein surface.
Journal of Biomolecular NMR, 1996
In this communication a new NMR experiment for the safe observation and quantification of waterprotein exchange phenomena is presented. It combines a water-selective pulse, offering chemical shiftbased separation, and the off-resonance ROESY dynamic filter, which permits the elimination of the unwanted intramolecular dipolar cross relaxation of protein protons. Moreover, pulsed field gradients are used for the suppression of radiation damping and the solvent signal. The straightforward incorporation of this sequence in heteronuclear experiments is demonstrated for the case of the DNA-binding domain of the alcohol regulator protein.
Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR
Journal of Biomolecular Nmr - J BIOMOL NMR, 2001
Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contributi...
Water’s Variable Role in Protein Stability Uncovered by Liquid-Observed Vapor Exchange NMR
Biochemistry, 2021
Water is essential to protein structure and stability, yet our understanding of how water shapes proteins is far from thorough. Our incomplete knowledge of protein-water interactions is due in part to a long-standing technological inability to assess experimentally how water removal impacts local protein structure. It is now possible to obtain residue-level information on dehydrated protein structures via liquid-observed vapor exchange (LOVE) NMR, a solution NMR technique that quantifies the extent of hydrogen-deuterium exchange between unprotected amide protons of a dehydrated protein and D 2 O vapor. Here, we apply LOVE NMR, Fourier transform infrared spectroscopy, and solution hydrogen-deuterium exchange to globular proteins GB1, CI2, and two variants thereof to link mutation-induced changes in the dehydrated protein structure to changes
Critical Reviews in Biochemistry and Molecular Biology, 1989
I. INTRODUCTION* Over the last decade, modem molecular biology, and in particular recombinant DNA technology, has had a major impact on a large number of biological disciplines. If one considers the area of protein biochemistry, for example, the number of protein sequences that has become available has reached over lO,OOO, the majority being derived from the nucleotide sequences of their respective genes. While this wealth of data are clearly impressive, amino acid sequences per se are of limited value in understanding protein function. In this respect, it is essential to determine the three-dimensional (3D) structures of proteins, and use this structural information in conjunction with functional data to study catalysis, ligand binding, assembly, and other processes in which proteins play a key role. Until recently, the only experimental technique available for determining 3D protein structures has been single crystal X-ray diffraction. One of the rate-limiting factors in solving an X-ray structure lies not only in obtaining suitable crystals that diffract to sufficient resolution, but also appropriate heavy atom derivatives to determine the phases of the reflections accurately. Consequently, it is not suprising that the number of protein X-ray structures solved to date (a few hundred) is several orders of magnitude smaller than the number of available proteins sequences. Over the last few years a second method of determining protein structures has been developed that makes use of nuclear magnetic resonance (NMR) spectroscopy. Unlike crystallography, NMR measurements are carried out in solution under potentially physiological conditions and therefore are not hampered by the ability or the inability of a protein to crystallize. Although the field of macromolecular structure determination by NMR is still young, a reasonable number of NMR protein structures have already been published (Table 1). With more and more proteins being produced by recombinant DNA methods, there is little doubt that this number will rapidly increase. At the present time, however, the application of NMR to protein structure determination is limited to proteins up to M,-20,000, a restriction that may not be as severe as it might appear at first glance. It is known, for example, from protein structures solved by X-ray crystallography that the size of protein domains generally lies between 10 and 20 kDa. Such domains are therefore clearly amenable to the NMR approach. Thus, it is likely that the expression and production of protein domains by recombinant DNA technology will make larger proteins accessible to NMR structure determination by studying their functional domains one by one. * A list of abbreviations used throughout this article appears following the text.
Journal of Molecular Biology, 1998
The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2 H and 17 O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 C. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/ operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/ operator complexes shows very similar structural environments for the water molecules mediating speci®c contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition.
Ordering effect of protein surfaces on water dynamics: NMR relaxation study
Biophysical Chemistry, 2019
Proteins in solution affect the structural and dynamic properties of the bulk water at the proteinwater interface, resulting in a contribution to the order of the hydration water. Theoretical and experimental NMR relaxation methods were developed to study the dynamic properties of water molecules in the protein hydration shell. Water non-selective and selective relaxation rates, were shown to be sensitive to contributions from ordered solvent molecules at protein surface. The average rotational correlation time of water molecules in the protein hydration shell was determined for three protein systems of different size: ribonuclease A, human serum albumin and fibrinogen. The knowledge of these properties is an important step towards the determination of the size of the water ordering contributions originate in proteins systems.