Fluorescence and FTIR study of pressure-induced structural modifications of horse liver alcohol dehydrogenase (HLADH).

The process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native-like state was assumed. Higher pressures (100-400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule-like state was proposed. Further increase of pressure (400-600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure-induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.     

High pressure NMR studies of hemoproteins. The effect of pressure on the quaternary structure of hemoglobin

Proton NMR spectra for nitrosyl-, aquomet- and deoxy des-Arg(α141)-hemoglobin in H₂O were studied at high pressures up to 1400 atm with attention to the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds. For aquomethemoglboin, the T state marker signal at 6.4 ppm is insensitive to pressure while the R state marker signal at 6.0 ppm exhibits progressive upfield shift upon pressurization. For nitrosylhemoglobin, the T state signals at 9.6 and 6.5 ppm decrease their intensities upon pressurization while the R state marker signal at 6.0ppm remains unchanged. Pressure-induced spectral changes for some of exchangeable resonances are also encountered for deoxy des-Arg(α141)-hemoglobin while the R and T quaternary structural indicators at 6.0 and 9.4 ppm are insensitive to pressure. These pressure-induced spectral changes for these hemoglobin derivatives are significantly distinguished from those associated with the R-T transition induced by addition of IHP or by variatiuon of pH. It is therefore concluded that pressure induces subtle quaternary structural changes in these hemoglobin derivatives without causing the R-T transition.     

Combined Low Calcium and Lack Magnesium Is a Risk Factor for Motor Deficit in Mice

To clarify the mechanism of pressure-induced meat tenderization or acceleration of meat conditioning, the pressure-induced morphological and biochemical changes in sarcoplasmic reticulum (SR), and Ca^{2 +} release from SR in the rabbit skeletal muscle treated with high pressure (100–300 MPa, 5min) were investigated in comparison with those of the SR from conditioned muscle.

The destruction of the membrane structure of the SR expanded with increasing pressure applied to the muscle. Significant changes in the SDS–PAGE profile were not observed in the SR from the pressurized muscle up to 200 MPa, but a marked decrease of the ATPase protein and high-affinity Ca²⁺-binding protein were observed in the SR from the pressurized muscle at 300 MPa. The ATPase activities increased in the SR isolated from the muscle exposed to high pressure up to 200 MPa. When the muscle was pressurized at 300 MPa, the ATPase activity dropped to the same level with that of the SR from the untreated muscle.Ca²⁺ uptake ability of the SR vesicles measured using a fluorescent chelating reagent decreased with increasing pressure applied to the muscle. Ultrastructural studies showed that Ca²⁺, which was mainly localized in the SR region of the untreated fiber bundles, was translocated into myofibrillar space in the pressurized muscle.

It is clear that a brief exposure of the muscle to high pressure causes considerable changes in membrane structure and biochemical function of SR as compared with those of SR in the muscle induced by conditioning.

The pressure-induced Ca^{2 +} release and loss of the structural regularity of myofibrils may be one of the causes for meat tenderization and acceleration of meat conditioning induced by high-pressure treatment.     

15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure

The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa(-1) at 278 K and 2.20 ppm GPa(-1), respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa(-1). Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the beta-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded beta-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.     

An improved ultrafast 2D NMR experiment: Towards atom-resolved real-time studies of protein kinetics at multi-Hz rates

Multidimensional NMR spectroscopy is a well-established technique for the characterization of structure and fast-time-scale dynamics of highly populated ground states of biological macromolecules. The investigation of short-lived excited states that are important for molecular folding, misfolding and function, however, remains a challenge for modern biomolecular NMR techniques. Off-equilibrium real-time kinetic NMR methods allow direct observation of conformational or chemical changes by following peak positions and intensities in a series of spectra recorded during a kinetic event. Because standard multidimensional NMR methods required to yield sufficient atom-resolution are intrinsically time-consuming, many interesting phenomena are excluded from real-time NMR analysis. Recently, spatially encoded ultrafast 2D NMR techniques have been proposed that allow one to acquire a 2D NMR experiment within a single transient. In addition, when combined with the SOFAST technique, such ultrafast experiments can be repeated at high rates. One of the problems detected for such ultrafast protein NMR experiments is related to the heteronuclear decoupling during detection with interferences between the pulses and the oscillatory magnetic field gradients arising in this scheme. Here we present a method for improved ultrafast data acquisition yielding higher signal to noise and sharper lines in single-scan 2D NMR spectra. In combination with a fast-mixing device, the recording of ¹H–¹⁵N correlation spectra with repetition rates of up to a few Hertz becomes feasible, enabling real-time studies of protein kinetics occurring on time scales down to a few seconds.     

NMR assignments of PI3-SH3 domain aided by protonless NMR spectroscopy

We report here the near complete assignments of native bovine PI3-SH3 domain, which has been a model system for protein folding, misfolding and amyloid fibril formation. The use of ¹³C-detected protonless NMR spectroscopy is instrumental in assigning the spin system of the proline residue at the C-terminus in addition to the missing resonances in proton-based NMR spectra due to rapid solvent exchange. It also helps assign the resonances of all three proline amine nitrogen nuclei, which are underrepresented in the database. Comparison of the backbone ¹³C resonances of PI3-SH3 in its native and amyloid fibril states shows that the aggregation of PI3-SH3 is accompanied by major conformational rearrangements.     

NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferredoxin protein from Desulphovibrio gigas, FdI, FdI′, and FdII was carried out. FdI and FdI′ are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted resonances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the “three state hypothesis” terminology it is shown that FdI_ox is predominantly in the C^2? state and changes upon reduction into the C^3? state, while FdII_ox is in the C^? state and changes into the C^2? state. FdI′ does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp.. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.     

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state ²H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific ²H labels have been introduced into the methyl groups of retinal and solid-state ²H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent ²H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu¹²² and Trp²⁶⁵ within the binding pocket. The solid-state ²H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.     

Structural stability of human butyrylcholinesterase under high hydrostatic pressure

Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200 MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700 MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400 ± 50 MPa. Addition of either 0.1 M ammonium sulphate (a kosmotropic salt) or 0.1 M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.     

Measurement of protein backbone <Superscript>13</Superscript>CO and <Superscript>15</Superscript>N relaxation dispersion at high resolution

The ³¹P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the ³¹P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH₂p and GTPγS was measured in the absence and presence of Mg²⁺-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B ₁ was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg²⁺·GMP and Mg²⁺·GppNHp the second order pressure coefficients are positive. To describe the data of Mg²⁺·GppCH₂p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg²⁺ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure ³¹P NMR data on free Mg²⁺-GDP and Mg²⁺-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.     

Modeling of Hidden Structures Using Sparse Chemical Shift Data from NMR Relaxation Dispersion

NMR relaxation dispersion measurements report on conformational changes occurring on the μs-ms timescale. Chemical shift information derived from relaxation dispersion can be used to generate structural models of weakly populated alternative conformational states. Current methods to obtain such models rely on determining the signs of chemical shift changes between the conformational states, which are difficult to obtain in many situations. Here, we use a “sample and select” method to generate relevant structural models of alternative conformations of the C-terminal-associated region of Escherichia coli dihydrofolate reductase (DHFR), using only unsigned chemical shift changes for backbone amides and carbonyls (¹H, ¹⁵N, and ¹³C′). We find that CS-Rosetta sampling with unsigned chemical shift changes generates a diversity of structures that are sufficient to characterize a minor conformational state of the C-terminal region of DHFR. The excited state differs from the ground state by a change in secondary structure, consistent with previous predictions from chemical shift hypersurfaces and validated by the x-ray structure of a partially humanized mutant of E. coli DHFR (N23PP/G51PEKN). The results demonstrate that the combination of fragment modeling with sparse chemical shift data can determine the structure of an alternative conformation of DHFR sampled on the μs-ms timescale. Such methods will be useful for characterizing alternative states, which can potentially be used for in silico drug screening, as well as contributing to understanding the role of minor states in biology and molecular evolution.     

Solid state NMR sequential resonance assignments and conformational analysis of the 2×10.4 kDa dimeric form of the Bacillus subtilis protein Crh

Solid state NMR sample preparation and resonance assignments of the U-[¹³C,¹⁵N] 2×10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra– and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all ¹⁵N, ¹³C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.     

Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired ¹³C and ¹⁵N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and α/β-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle.     

Characterization of low-lying excited states of proteins by high-pressure NMR

Hydrostatic pressure alters the free energy of proteins by a few kJ mol⁻¹, with the amount depending on their partial molar volumes. Because the folded ground state of a protein contains cavities, it is always a state of large partial molar volume. Therefore pressure always destabilises the ground state and increases the population of partially and completely unfolded states. This is a mild and reversible conformational change, which allows the study of excited states under thermodynamic equilibrium conditions. Many of the excited states studied in this way are functionally relevant; they also seem to be very similar to kinetic folding intermediates, thus suggesting that evolution has made use of the ‘natural’ dynamic energy landscape of the protein fold and sculpted it to optimise function. This includes features such as ligand binding, structural change during the catalytic cycle, and dynamic allostery.     

A methyl <Superscript>1</Superscript>H double quantum CPMG experiment to study protein conformational exchange

Protein conformational changes play crucial roles in enabling function. The Carr–Purcell–Meiboom–Gill (CPMG) experiment forms the basis for studying such dynamics when they involve the interconversion between highly populated and sparsely formed states, the latter having lifetimes ranging from ~?0.5 to ~?5 ms. Among the suite of experiments that have been developed are those that exploit methyl group probes by recording methyl ¹H single quantum (Tugarinov and Kay in J Am Chem Soc 129:9514–9521, 2007) and triple quantum (Yuwen et al. in Angew Chem Int Ed Engl 55:11490–11494, 2016) relaxation dispersion profiles. Here we build upon these by developing a third experiment in which methyl ¹H double quantum coherences evolve during a CPMG relaxation element. By fitting single, double, and triple quantum datasets, akin to recording the single quantum dataset at static magnetic fields of B_o, 2B_o and 3B_o, we show that accurate exchange values can be obtained even in cases where exchange rates exceed 10,000 s^?1. The utility of the double quantum experiment is demonstrated with a pair of cavity mutants of T4 lysozyme (T4L) with ground and excited states interchanged and with exchange rates differing by fourfold (~?900 s^?1 and ~?3600 s^?1), as well as with a fast-folding domain where the unfolded state lifetime is ~?80 µs.     

Hyperfine shift NMR studies of hydrated phospholipid inverted micelles

The ³¹P nuclear magnetic resonance (NMR) spectra of benzene solutions of hydrated dipalmitoyl lecithin (DPL) inverted micelles, with and without incorporated paramagnetic lanthanide ions, have been recorded. Individual resonances for micelles containing none, one, and two ions can be resolved and observed in the presence of one another. The relative intensities of these peaks yield some information on the state of aggregation of lipid inverted micelles prepared by ultrasonic irradiation. The relative intensities and chemical shifts of resonances of unsonicated mixtures of preformed micelles containing different numbers of ions per micelle indicate that some kind of equilibration occurs. The data are consistent with a selective fusion of multi-ion micelles with ion-free micelles. The NMR spectra place constraints on the lifetimes of metal ions and lipid and water molecules within a micelle before transfer to another.     

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the ¹H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.     

Relating side-chain mobility in proteins to rotameric transitions: Insights from molecular dynamics simulations and NMR

The dynamic aspect of proteins is fundamental to understanding protein stability and function. One of the goals of NMR studies of side-chain dynamics in proteins is to relate spin relaxation rates to discrete conformational states and the timescales of interconversion between those states. Reported here is a physical analysis of side-chain dynamics that occur on a timescale commensurate with monitoring by ²H spin relaxation within methyl groups. Motivated by observations made from tens-of-nanoseconds long MD simulations on the small protein eglin c in explicit solvent, we propose a simple molecular mechanics-based model for the motions of side-chain methyl groups. By using a Boltzmann distribution within rotamers, and by considering the transitions between different rotamer states, the model semi-quantitatively correlates the population of rotamer states with ‘model-free’ order parameters typically fitted from NMR relaxation experiments. Two easy-to-use, analytical expressions are given for converting S²_axis’ values (order parameter for C–CH₃ bond) into side-chain rotamer populations. These predict that S²_axis’ values below 0.8 result from population of more than one rotameric state. The relations are shown to predict rotameric sampling with reasonable accuracy on the ps–ns timescale for eglin c and are validated for longer timescales on ubiquitin, for which side-chain residual dipolar coupling (RDC) data have been collected.     

Fluorescent probe and NMR studies of the aggregation of bile salts in aqueous solution

The binding of the fluorescent probes 1-anilino-8-naphthalene sulfonate and dansyl cadaverine to the sodium salts of cholic, deoxycholic and dehydrocholic acids has been investigated. Enhanced probe solubilisation accompanies aggregation. Monitoring of fluorescence intensities as a function of bile salt concentration permits the detection of primary micelle formation, as well as secondary association. The transition concentrations obtained by fluorescence are in good agreement with values determined for the critical micelle concentrations, by other methods. Differences in the behaviour of cholate and deoxycholate have been noted. Fluorescence polarisation studies of 1,6-diphenyl-1,3,5-hexatriene solubilised in bile salt micelles suggest a higher microviscosity for the interior of the deoxycholate micelle as compared to cholate. ¹H NMR studies of deoxycholate over the range 1–100 mg/ml suggest that micelle formation leads to a greater immobilisation of the C₁₈ and C₁₉ methyl groups as compared to the C₂₁ methyl group. Well resolved ¹³C resonances are observed for all three steroids even at high concentration. Both fluorescence and NMR studies confirm that dehydrocholate does not aggregate.