Characterisation of denatured states of sensory rhodopsin II by solution-state NMR

Sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis, is a novel model system for membrane protein folding studies. Recently, the SDS-denatured states and the kinetics for reversible unfolding of pSRII have been investigated, opening the door to the first detailed characterisation of denatured states of a membrane protein by solution-state nuclear magnetic resonance (NMR) using uniformly ¹⁵N-labelled pSRII. SDS denaturation and acid denaturation of pSRII both lead to fraying of helix ends but otherwise small structural changes in the transmembrane domain, consistent with little changes in secondary structure and disruption of the retinal-binding pocket and tertiary structure. Widespread changes in the backbone amide dynamics are detected in the form of line broadening, indicative of μs-to-ms timescale conformational exchange in the transmembrane region. Detailed analysis of chemical shift and intensity changes lead to high-resolution molecular insights on structural and dynamics changes in SDS- and acid-denatured pSRII, thus highlighting differences in the unfolding pathways under the two different denaturing conditions. These results will form the foundation for furthering our understanding on the folding and unfolding pathways of retinal-binding proteins and membrane proteins in general, and also for investigating the importance of ligand-binding in the folding pathways of other ligand-binding membrane proteins, such as GPCRs.     

Measurement of chemical exchange between RNA conformers by F NMR

Many noncoding RNA molecules adopt alternative secondary and tertiary conformations that are critical for their roles in gene expression. Although many of these rearrangements are mediated by other biomolecular components, it is important to evaluate the equilibrium relationship of the conformers. To measure the spontaneous interconversion in a bi-stable RNA stem loop sequence into which a single ¹⁹F-uridine label was incorporated, a ¹⁹F–¹⁹F EXSY experiment was employed. The kinetic exchange rate measured from EXSY experiments for this system was 37.3 ± 2.8 s⁻¹. The advantage of this approach is that exchange kinetics can be monitored in any RNA sequence into which a single ¹⁹F nucleotide is incorporated by commercial synthesis. This method is therefore suitable for application to biologically significant systems in which dynamic conformational rearrangement is important for function and may therefore facilitate studies of RNA structure–function relationships.     

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.     

Cellular uptake of soluble and aggregated ferritin: distinction between pinocytosis and phagocytosis.

Cellular uptake of ferritin amounting to 0-5 mug/mg cell protein or more can be measured colorimetrically on the basis of ferritin-iron content. 131I-serum albumin, soluble ferritin and aggregated ferritin used in equimolar concentrations are taken up differently by Sarcoma SI80 cells in culture. The net uptakes in 2 h at 37 degrees C are 0-065, 4-3 and 24-7 mug/mg cell protein or 0-93, 8-0 and 45-7 mumol, respectively. Albumin uptake is not inhibited by a 26-fold molar ferritin excess but is significantly inhibited by a 43-fold excess. The transport mechanism of the ferritins differs from that of albumin in that it is significantly inhibitable by 2 times 10(-4) M monoiodoacetate. Soluble ferritin contains small aggregates which are removed by filtration through Millipore membranes of 0-05, 0-1 and 0-22 mum. When the 0-1-mum filtrate is re-examined, uptake is no longer inhibited by iodoacetate. Since it can be inferred from other work that albumin is taken up by pinocytosis and ferritin aggregates by phagocytosis, the difference in susceptibility to inhibition is proposed as a way to distinguish pinocytosis from phagocytosis. Ferritin may form larger visible aggregates in culture medium. The transport mechanism of this aggregated ferritin differs from that of soluble unfiltered ferritin in that it causes concomitant enhancement of albumin uptake. Albumin transported by virtue of this effect becomes partially susceptible to iodoacetate. Thus, in addition to a distinction between pinocytosis and phagocytosis, our data single out 2 forms of albumin transport and 3 forms of ferritin transport.     

基于氢氘交换核磁共振技术研究蛋白质与阳离子交换介质的相互作用

原位实时捕捉多孔介质内的蛋白结构变化信息是蛋白质层析失活机理研究中的难点。为此,本文发展了蛋白质氢氘交换与核磁共振(Nuclear magnetic resonance,NMR)相结合的新型蛋白质液固界面表征路线。研究了溶菌酶在溶液态以及在阳离子交换介质内部吸附态时的去折叠行为,并揭示了蛋白与阳离子交换介质(SP Sepharose FF)相互作用机理。溶液态溶菌酶的一维核磁共振氢谱动力学显示蛋白质去折叠可导致残基暴露进而加快氢氘交换速率。吸附态溶菌酶的二维氢-氢全相关谱图(Total correlation spectroscopy,TOCSY)以及残基峰强度显示,溶菌酶在吸附态时的去折叠呈区域性,无规则卷曲(Coil,bend,and turn)片段的酰胺氢信号更容易失去,而二级结构域(α-helix,β-sheet)对酰胺氢信号保护更好。最终,利用蛋白表面静电势模拟计算结合氢氘标记的蛋白核核磁数据可确定出溶菌酶与阳离子交换介质的作用位点。这对于深刻理解层析过程中蛋白与层析介质微观作用机理以及层析过程中吸附剂的选择、设计具有重要意义,也为获取蛋白质与生物材料之间相互作用研究提供新的有效工具。     

Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR.

The extent of deuterium labeling of hen lysozyme, its three-disulfide derivative, and the homologous alpha-lactalbumins, has been measured by both mass spectrometry and NMR. Different conformational states of the proteins were produced by varying the solution conditions. Alternate protein conformers were found to contain different numbers of 2H atoms. Furthermore, measurement in the gas phase of the mass spectrometer or directly in solution by NMR gave consistent results. The unique ability of mass spectrometry to distinguish distributions of 2H atoms in protein molecules is exemplified using samples prepared to contain different populations of 2H-labeled protein. A comparison of the peak widths of bovine alpha-lactalbumin in alternate solution conformations but containing the same average number of 2H atoms showed dramatic differences due to different 2H distributions in the two protein conformers. Measurement of 2H distributions by ESI-MS enabled characterization of conformational averaging and structural heterogeneity. In addition, a time course for hydrogen exchange was examined and the variation in distributions of 2H atom compared with simulations for different hydrogen exchange models. The results clearly show that exchange from the native state of bovine alpha-lactalbumin at 15 degrees C is dominated by local unfolding events.     

Characterization of the interaction between lysyl-tRNA synthetase and laminin receptor by NMR

Lysyl-tRNA synthetase (KRS) interacts with the laminin receptor (LR/RPSA) and enhances laminin-induced cell migration in cancer metastasis. In this nuclear magnetic resonance (NMR)-based study, we show that the anticodon-binding domain of KRS binds directly to the C-terminal region of 37LRP, and the previously found inhibitors BC-K-01 and BC-K-YH16899 interfere with KRS–37LRP binding. In addition, the anticodon-binding domain of KRS binds to laminin, observed by NMR and SPR. These results provide crucial insights into the structural characteristics of the KRS–LR interaction on the cell surface.     

Subunit exchange between membranous and soluble forms of bovine dopamine beta-hydroxylase

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms: soluble and membrane-bound. In a previous study, it was shown that the tetrameric, soluble form of the enzyme undergoes dissociation into two identical dimeric subunits and that this subunit dissociation is dependent on pH and ADP binding (Dhawan, S., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1986) J. Biol. Chem. 261, 7680-7684). Here we report the effect of pH and ADP on the dissociation of the membranous form of dopamine beta-hydroxylase into two nonidentical subunits. Negative stain electron microscopy of purified membranous hydroxylase showed largely tetrameric species together with occasional dimeric species. The tetrameric images of membranous hydroxylase were similar to, but clearly different from, previously published negative stain images of soluble hydroxylase (Duong, L. T., Fleming, P. J., and Ornberg, R. L. (1985) J. Biol. Chem. 260, 2393-2398). Quantitative binding of ADP to the membranous hydroxylase revealed the existence of two binding sites per dimeric subunit. ADP binding and low pH both promote dissociation of a hydrophilic, catalytically active subunit from the membranous enzyme reconstituted onto phospholipid vesicles. Kinetic analyses of reconstituted membranous hydroxylase activity were consistent with the existence of tetrameric and dimeric catalytic species in equilibrium. All of the hydrophilic subunits of the purified soluble hydroxylase bind to the hydrophobic subunits of the reconstituted membranous hydroxylase. We propose that, in the chromaffin granules, the soluble hydroxylase subunits are in equilibrium association with the membrane-bound hydroxylase subunits and that the hydrophilic subunits of both soluble and membranous hydroxylase are identical.     

Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated (13)C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (13)CO experiments however the large separation between (13)CO and (13)C(alpha) chemical shifts offers hope that robust (13)CO dispersion profiles can be recorded on uniformly (13)C labeled samples, leading to the extraction of accurate (13)CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-(13)C]-pyruvate and NaH(13)CO(3,) or uniformly labeled, generated from (13)C-glucose. Very similar (13)CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.     

NMR measurements of proton exchange between solvent and peptides and proteins

Scope and limitations of the NMR based methods, equilibration and magnetization transfer, for measuring proton exchange rates of amide protons in peptides and proteins with water protons are discussed. Equilibration is applied to very slow processes detected by hydrogen-deuterium exchange after a solute is dissolved in D2O. Magnetization transfer allows to study moderately rapid processes in H2O. A number of precautions should be undertaken in order to avoid systemic errors inherent in the magnetization transfer method.     

Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a β-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the β-sheet conformation (∼ 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (> 40%), the β-sheet structures were gradually changed to helical conformations and the relative content of the helical and β-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the β-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.     

Starch phosphorylation--maltosidic restrains upon 3'- and 6'-phosphorylation investigated by chemical synthesis, molecular dynamics and NMR spectroscopy

Phosphorylation is the only known in vivo substitution of starch, yet no structural evidence has been provided to explain its implications of the amylosidic backbone and its stimulating effects on starch degradation in plants. In this study, we provide evidence for a major influence on the glucosidic bond in starch specifically induced by the 3-O-phosphate. Two phosphorylated maltose model compounds were synthesized and subjected to combined molecular dynamics (MD) studies and 950 MHz NMR studies. The two phosphorylated disaccharides represent the two possible phosphorylation sites observed in natural starches, namely maltose phosphorylated at the 3'- and 6'-position (maltose-3'-O-phosphate and maltose-6'-O-phosphate). When compared with maltose, both of the maltose-phosphates exhibit a restricted conformational space of the alpha(1-->4) glycosidic linkage. When maltose is phosphorylated in the 3'-position, MD and NMR show that the glucosidic space is seriously restricted to one narrow potential energy well which is strongly offset from the global potential energy well of maltose and almost 50 degrees degrees from the Phi angle of the alpha-maltose crystal structure. The driving force is primarily steric, but the configuration of the structural waters is also significantly altered. Both the favored conformation of the maltose-3'-phosphate and the maltose-6'-phosphate align well into the 6-fold double helical structure of amylopectin when the effects on the glucosidic bond are not taken into account. However, the restrained geometry of the glucosidic linkage of maltose-3'-phosphate cannot be accommodated in the helical structure, suggesting a major local disturbing effect, if present in the starch granule semi-crystalline lattice.     

Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr–Purcell–Meiboom–Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k _ex = 500–2000 s⁻¹ were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83–87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local ‘breathing’ motions rather than a concerted global conformational change. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of the interactions between coumarin-derivatives and acetylcholinesterase: Examination by NMR and docking simulations

Alzheimer’s disease (AD) is one of the most common forms of dementia and a significant threat to the elderly populations, especially in the Western world. The rapid hydrolysis of the principal neurotransmitter into choline and acetate by acetylcholinesterase (AChE) at synapses causes the loss of cognitive response that becomes the real cause of AD. Therefore, inhibition of AChE is the most fundamental therapy among currently available treatments for AD. In this context, we designed and performed molecular recognitions studies of coumarin-based inhibitors towards AChE. STD NMR and Tr-NOESY applications were utilized to evaluate the binding epitope, the dissociation constant (K_D) and bound conformations of these inhibitors within this inhibitor-AChE complex. Compound 1, which has a similar inhibition activity to tacrine (a current drug) led in this study as a stronger binder with K_D?=?30 μM ,even greater than tacrine (K_D?=?140 μM). Moreover, docking simulations mimic NMR results and provided evidence of synchronizing binding of compound 1 with three sites; the peripheral anionic site, the bottom of the gorge, and the catalytic site. Therefore, we envisioned from our experimental and theoretical results that coumarin-based inhibitors containing a piperidinyl scaffold might be a potential drug candidates for AD in the future.     

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a 13C-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-13C]glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using 1H spin-echo NMR, while 13C[1H-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.     

Characterization of the near native conformational states of the SAM domain of Ste11 protein by NMR spectroscopy

The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen‐activated protein kinase cascades. In the current study, urea‐induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions. Proteins 2014; 82:2957–2969. © 2014 Wiley Periodicals, Inc.