Hydrogen bonds are key constituents of biomolecular structures, and their response to external perturbations may reveal important insights about the most stable components of a structure. NMR spectroscopy can probe hydrogen bond deformations at very high resolution through hydrogen bond scalar couplings (HBCs). However, the small size of HBCs has so far prevented a comprehensive quantitative characterization of protein hydrogen bonds as a function of the basic thermodynamic parameters of pressure and temperature. Using a newly developed pressure cell, we have now mapped pressure- and temperature-dependent changes of 31 hydrogen bonds in ubiquitin by measuring HBCs with very high precision. Short-range hydrogen bonds are only moderately perturbed, but many hydrogen bonds with large sequence separations (high contact order) show greater changes. In contrast, other high-contact-order hydrogen bonds remain virtually unaffected. The specific stabilization of such topologically important connections may present a general principle with which to achieve protein stability and to preserve structural integrity during protein function.
Proteins denature not only at high, but also at low temperature as well as high pressure. These denatured states are not easily accessible for experiment, because usually heat denaturation causes aggregation, whereas cold or pressure denaturation occurs at temperatures well below the freezing point of water or pressures above 5 kbar, respectively. Here we have obtained atomic details of the pressure-assisted, cold-denatured state of ubiquitin at 2,500 bar and 258 K by high-resolution NMR techniques. Under these conditions, a folded, native-like and a disordered state exist in slow exchange. Secondary chemical shifts show that the disordered state has structural propensities for a native-like N-terminal β-hairpin and α-helix and a nonnative C-terminal α-helix. These propensities are very similar to the previously described alcohol-denatured (A-)state. Similar to the A-state, 15N relaxation data indicate that the secondary structure elements move as independent segments. The close similarity of pressure-assisted, cold-denatured, and alcohol-denatured states with native and nonnative secondary elements supports a hierarchical mechanism of folding and supports the notion that similar to alcohol, pressure and cold reduce the hydrophobic effect. Indeed, at nondenaturing concentrations of methanol, a complete transition from the native to the A-state can be achieved at ambient temperature by varying the pressure from 1 to 2,500 bar. The methanol-assisted pressure transition is completely reversible and can also be induced in protein G. This method should allow highly detailed studies of protein-folding transitions in a continuous and reversible manner.protein unfolding | thermodynamics | protein dynamics | heteronuclear NMR I t has long been known that proteins unfold not only at high temperatures, but also at high pressures (1) as well as low temperatures (2). The so-called heat, pressure, and cold denaturations can be described in a unified way by Hawley's theory (1,3). This theory assumes a simplified two-state model of protein unfolding, where the free energy difference between folded and unfolded states is a general parabolic function of temperature and pressure:In Eq. 1 Δ indicates the difference of the respective value between the denatured and the native state; β is the compressibility factor ð∂V =∂ pÞ T ; α is the thermal expansivity factor ð∂V =∂TÞ p ; C p is the heat capacity Tð∂S=∂TÞ p ; and p 0 , T 0 is an arbitrarily chosen reference point. The phase boundary between denatured and native states is then given by the condition ΔG = 0, which corresponds to a tilted ellipse within the pT plane for commonly observed values of Δβ < 0, ΔC p > 0, and Δα > 0. This two-state model is clearly an oversimplification, because both folded and unfolded states can be heterogeneous, and due to the paucity of data it is also unclear whether the heat-, cold-, and pressuredenatured states are identical. Nevertheless the model presents a valuable general framework to pinpoint the main contributing thermodynamic entities, which...
A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]N hydrogen bonds (H-bonds) in 15N isotope-labeled biomolecules. This quantitative HNN-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect magnetically active (15)N nuclei of the H-bond donor and acceptor. Detectable H-bonds comprise the imino H-bonds in canonical Watson-Crick base pairs, many H-bonds in unusual nucleic acid base pairs and H-bonds between protein backbone or side-chain N-H donor and N acceptor moieties. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor in a single experiment. The size of the scalar couplings can be related to H-bond geometries and as a time average to H-bond dynamics. The time required to detect the H-bonds is typically less than 1 d at millimolar concentrations for samples of molecular weight < or = approximately 25 kDa. A C15N/13C-labeled potato spindle tuber viroid T1 RNA domain is used as an example to illustrate this procedure.
A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]O=C hydrogen bonds (H-bonds) in 15N and 13C isotope-labeled biomolecules. This quantitative 'long-range' HNCO-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect the magnetically active (15)N and (13)C nuclei on both sides of the H-bond. Detectable H-bonds comprise the canonical backbone H-bonds in proteins as well as other H-bonds in proteins and nucleic acids with N-H donors and O=C (carbonylic or carboxylic) acceptors. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor, in a single experiment. The size of the scalar couplings can be related to H-bond geometries. The time required to detect the N-H[...]O=C H-bonds in small proteins (< or = approximately 10 kDa) is typically on the order of 1 d at millimolar concentrations, whereas H-bond detection for larger proteins (< or = approximately 30 kDa) may be possible within several days depending on concentration, isotope composition, magnetic field strength and molecular weight. The proteins ubiquitin (8.6 kDa), dimeric RANTES (2 x 8.5 kDa) and MAP30 (30 kDa) are used as examples to illustrate this procedure.
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