Off-resonance 13C–2H REDOR NMR for site-resolved studies of molecular motion

We introduce a ¹³C–²H Rotational Echo DOuble Resonance (REDOR) technique that uses the difference between on-resonance and off-resonance ²H irradiation to detect dynamic segments in deuterated molecules. By selectively inverting specific regions of the ²H magic-angle spinning (MAS) sideband manifold to recouple some of the deuterons to nearby carbons, we distinguish dynamic and rigid residues in 1D and 2D ¹³C spectra. We demonstrate this approach on deuterated GB1, H/D exchanged GB1, and perdeuterated bacterial cellulose. Numerical simulations reproduce the measured mixing-time and ²H carrier-frequency dependence of the REDOR dephasing of bacterial cellulose. Combining numerical simulations with experiments thus allow the extraction of motionally averaged quadrupolar couplings from REDOR dephasing values.

     

Evidence for high molecular weight Na−Ca exchange in cardiac sarcolemmal vesicles

Summary Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na–Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na–Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (M _r) of approximately 226,000±20,000sd (n=6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 11 with mobile phase buffer containing 50mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na–Ca exchange activity. Na–Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na–Ca exchange activity were 91±7 and 68±11%, respectively. In the peak fraction, Na–Ca exchange specific activity increased two-to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimumM _r of 224,000 Da. Specific⁴⁵Ca²⁺-binding SL proteins withM _r of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose.These data suggest that native cardiac Na–Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na–Ca exchange protein(s) remains incomplete.     

Utilization of lysine 13C-methylation NMR for protein–protein interaction studies

Chemical modification is an easy way for stable isotope labeling of non-labeled proteins. The reductive ¹³C-methylation of the amino group of the lysine side-chain by ¹³C-formaldehyde is a post-modification and is applicable to most proteins since this chemical modification specifically and quickly proceeds under mild conditions such as 4 °C, pH 6.8, overnight. ¹³C-methylation has been used for NMR to study the interactions between the methylated proteins and various molecules, such as small ligands, nucleic acids and peptides. Here we applied lysine ¹³C-methylation NMR to monitor protein–protein interactions. The affinity and the intermolecular interaction sites of methylated ubiquitin with three ubiquitin-interacting proteins were successfully determined using chemical-shift perturbation experiments via the ¹H–¹³C HSQC spectra of the ¹³C-methylated-lysine methyl groups. The lysine ¹³C-methylation NMR results also emphasized the importance of the usage of side-chain signals to monitor the intermolecular interaction sites, and was applicable to studying samples with concentrations in the low sub-micromolar range.     

Folding of an all-helical Greek-key protein monitored by quenched-flow hydrogen–deuterium exchange and NMR spectroscopy

To advance our understanding of the protein folding process, we use stopped-flow far-ultraviolet (far-UV) circular dichroism and quenched-flow hydrogen–deuterium exchange coupled with nuclear magnetic resonance (NMR) spectroscopy to monitor the formation of hydrogen-bonded secondary structure in the C-terminal domain of the Fas-associated death domain (Fadd-DD). The death domain superfamily fold consists of six α-helices arranged in a Greek-key topology, which is shared by the all-β-sheet immunoglobulin and mixed α/β-plait superfamilies. Fadd-DD is selected as our model death domain protein system because the structure of this protein has been solved by NMR spectroscopy, and both thermodynamic and kinetic analysis indicate it to be a stable, monomeric protein with a rapidly formed hydrophobic core. Stopped-flow far-UV circular dichroism spectroscopy revealed that the folding process was monophasic and the rate is 23.4 s⁻¹. Twenty-two amide hydrogens in the backbone of the helices and two in the backbone of the loops were monitored, and the folding of all six helices was determined to be monophasic with rate constants between 19 and 22 s⁻¹. These results indicate that the formation of secondary structure is largely cooperative and concomitant with the hydrophobic collapse. This study also provides unprecedented insight into the formation of secondary structure within the highly populated Greek-key fold more generally. Additional insights are gained by calculating the exchange rates of 23 residues from equilibrium hydrogen–deuterium exchange experiments. The majority of protected amide protons are found on helices 2, 4, and 5, which make up core structural elements of the Greek-key topology.     

Identifying inter-residue resonances in crowded 2D 13C–13C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy

The feasibility of using difference spectroscopy, i.e. subtraction of two correlation spectra at different mixing times, for substantially enhanced resolution in crowded two-dimensional ¹³C–¹³C chemical shift correlation spectra is presented. With the analyses of ¹³C–¹³C spin diffusion in simple spin systems, difference spectroscopy is proposed to partially separate the spin diffusion resonances of relatively short intra-residue distances from the longer inter-residue distances, leading to a better identification of the inter-residue resonances. Here solid-state magic-angle-spinning NMR spectra of the full length M2 protein embedded in synthetic lipid bilayers have been used to illustrate the resolution enhancement in the difference spectra. The integral membrane M2 protein of Influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Based on known amino acid resonance assignments from amino acid specific labeled samples of truncated M2 sequences or from time-consuming 3D experiments of uniformly labeled samples, some inter-residue resonances of the full length M2 protein can be identified in the difference spectra of uniformly ¹³C labeled protein that are consistent with the high resolution structure of the M2 (22–62) protein (Sharma et al., Science 330(6003):509–512, 2010).     

13C-2H correlation NMR spectroscopy studies of the in vivo transformations of natural products from Artemisia annua

¹³C-²H correlation NMR spectroscopy (¹³C-²H COSY) permits the identification of ¹³C and ²H nuclei which are connected to one another by a single chemical bond via the sizeable ¹J_CD coupling constant. The practical development of this technique is described using a ¹³C-²H COSY pulse sequence which is derived from the classical ¹³C-¹H correlation experiment. An example is given of the application of ¹³C-²H COSY to the study of the biogenesis of natural products from the anti-malarial plant Artemisia annua, using a doubly-labelled precursor molecule. Although the biogenesis of artemisinin, the anti-malarial principle from this species, has been extensively studied over the past twenty years there is still no consensus as to the true biosynthetic route to this important natural product – indeed, some published experimental results are directly contradictory. One possible reason for this confusion may be the ease with which some of the metabolites from A. annua undergo spontaneous autoxidation, as exemplified by our recent in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid, and the application of ¹³C-²H COSY to this biosynthetic problem has been important in helping to mitigate against such processes. In this in vivo application of ¹³C-²H COSY, [15-¹³C²H₃]-dihydroartemisinic acid (the doubly-labelled analogue of the natural product from this species which was obtained through synthesis) was fed to A. annua plants and was shown to be converted into several natural products which have been described previously, including artemisinin. It is proposed that all of these transformations occurred via a tertiary hydroperoxide intermediate, which is derived from dihyroartemisinic acid. This intermediate was observed directly in this feeding experiment by the ¹³C-²H COSY technique; its observation by more traditional procedures (e.g., chromatographic separation, followed by spectroscopic analysis of the purified product) would have been difficult owing to the instability of the hydroperoxide group (as had been established previously by our in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid). This same hydroperoxide has been reported as the initial product of the spontaneous autoxidation of dihydroartemisinic acid in our previous in vitro studies. Its observation in this feeding experiment by the ¹³C-²H COSY technique, a procedure which requires the minimum of sample manipulation in order to achieve a reliable identification of metabolites (based on both ¹³C and ²H chemical shifts at the 15-position), provides the best possible evidence for its status as a genuine biosynthetic intermediate, rather than merely as an artifact of the experimental procedure.     

NMR studies on the monomer–tetramer transition of melittin in an aqueous solution at high and low temperatures

Melittin, a peptide of 26 amino acid residues, has been used as a model peptide for protein folding and unfolding, and extensive research has been done into its structure and conformational stability. Circular dichroism (CD) studies have demonstrated that melittin in an aqueous solution undergoes a transition from a helical tetramer to a random coil monomer not only by heating but also by cooling from room temperature (i.e., heat- and cold-denaturation, respectively). The heat-denaturation has been also examined by nuclear magnetic resonance (NMR) experiments, however, no NMR data have been presented on the cold-denaturation. In this paper, using proton ((1)H) NMR spectroscopy, we show that melittin undergoes conformational transitions from the monomer to the tetramer to the monomer by elevating temperature from 2 to 70 °C. Only melittin including a trans proline peptide bond participates in the transitions, whereas melittin including a cis proline one does not. The tetramer has maximum conformation stability at around 20 °C, and cooperativity of the heat-denaturation is extremely low.     

Is AKR2A an essential molecular chaperone for a class of membrane-bound proteins in plants?

The Arabidopsis ankyrin-repeat containing protein 2A (AKR2A) was shown to be an essential molecular chaperone for the peroxisomal membrane-bound ascorbate peroxidase 3 (APX3), because the biogenesis of APX3 depends on the function of AKR2A in plant cells. AKR2A binds specifically to a sequence in APX3 that is made up of a transmembrane domain followed by a few positively charged amino acid residues; this sequence is named as AKR2A-binding sequence or ABS. Interestingly, a sequence in the chloroplast outer envelope protein 7 (OEP7) shares similar features to ABS and is able to bind specifically to AKR2A, suggesting a possibility that proteins with a sequence similar to ABS could bind to AKR2A and they are all likely ligand proteins of AKR2A. This hypothesis was supported by analyzing five additional proteins that contain sequences similar to ABS using the yeast two-hybrid technique. A preliminary survey in the Arabidopsis genome indicates that there are at least 500 genes encoding proteins that contain sequences similar to ABS, which raises interesting questions: are these proteins AKR2A''s ligand proteins and does AKR2A play a critical role in the biogenesis of these proteins in plants?Key words: Arabidopsis, membrane protein, molecular chaperone, protein targeting, transmembrane domainThe Arabidopsis ankyrin-repeat containing protein 2A (AKR2A) is an essential molecular chaperone for the peroxisomal membrane-bound ascorbate peroxidase 3 (APX3).¹ Both AKR2A and APX3 were identified as GF14λ-interacting proteins^2,3 when the mode of action of a 14-3-3 protein, GF14λ⁴ was studied. In characterizing the enzymatic property of APX3, there was some initial difficulty in purifying the expressed APX3 from a bacterial expression system. Although APX3 could be expressed in E. coli cells in large quantities, as evidenced by directly boiling the bacterial cells and analyzing the bacterial cells by SDS-PAGE and Western blot analysis (Fig. 1), APX3 enzymatic activity in the supernatant fraction was not detectable after cells were broken by sonication (Fig. 1). The reason that APX3 activity was not detectable in the supernatant fraction was likely caused by the transmembrane domain that occurs at the C-terminal end of APX3; because these hydrophobic domains could interact with one another, forming insoluble aggregates in bacterial cells. When a truncated APX3 was expressed, i.e., APX3 without the transmembrane domain (APX3Δ in Fig. 1), APX3 activity was then detectable in the supernatant fraction of bacterial cellular extracts. If a protein is able to bind to APX3''s transmembrane domain immediately after or during translation of APX3, this protein could prevent APX3 from forming insoluble aggregates among themselves. APX3 activity would then be detectable in the supernatant fraction. Because some 14-3-3-interacting proteins were shown to interact with one another,⁵ the best candidate that could interact with APX3 should be AKR2A (because they are both GF14λ-interacting proteins). This possibility was tested by simultaneously expressing both APX3 and AKR2A in the same bacterial cell; APX3 activity was indeed detectable in the supernatant fraction of bacterial cellular extracts (Fig. 1).Open in a separate windowFigure 1Protein-protein interaction between AKR2A and APX3 in bacterial cells. (A) Analysis of APX3 activity in supernatant fractions of various bacterial cells. In lanes, APX3, supernatant from cells that express full-length APX3; APX3 + OMT 1, supernatant from cells that express both full-length APX3 and OMT 1 (O-methyltransferase1,⁷); APX3 + AKR2A, supernatant from cells that express both full-length APX3 and AKR2A; APX3Δ, supernatant from cells that express a partial APX3 (i.e., lacking the transmembrane domain and the last seven amino acid residues); APX3Δ + OMT 1, supernatant from cells that express both APX3Δ and OMT 1; APX3Δ + AKR2A, supernatant from cells that express both APX3Δ and AKR2A; OMT 1, supernatant from cells that express OMT1; AKR2A, supernatant from cells that express AKR2A. The white bands in the gel represent APX3 activities as assayed by using the method of Mittler and Zilinskas.⁸ (B) Bacterial cells expressing various target proteins were analyzed directly by using SDS-PAGE method and the positions of the expressed target proteins are marked on the right. (C) Bacterial cells expressing various target proteins were analyzed by western blot. The antibodies used are listed on the right.This was the first evidence that AKR2A interacts with APX3 and the interaction site involves the C-terminal transmembrane domain of APX3. To further define the amino acid residues involved in the AKR2A-APX3 interaction, yeast two-hybrid experiments were conducted with various deletion fragments of AKR2A and APX3.1 It was found that in addition to the transmembrane domain, the positively charged amino acid residues following the transmembrane domain also play a role in the AKR2A-APX3 interaction.¹ This sequence in APX3 was designated as AKR2A-binding sequence (ABS). In order to understand the biological function of the AKR2A-APX3 interaction, several akr2a mutants that displayed reduced or altered interaction with APX3 were created and analyzed. Results indicated that reduced AKR2A activity leads to severe developmental, phenotypic, and physiological abnormalities including reduced steady-state level of APX3 and reduced targeting of APX3 to peroxisomal membranes in Arabidopsis.¹ The pleiotropic nature of akr2a mutants indicated that AKR2A plays more roles in addition to chaperoning APX3. Indeed this work was corroborated by a finding that AKR2A is also required for the biogenesis of the chloroplast outer envelope protein 7 (OEP7).⁶ More importantly, the interaction between AKR2A and OEP7 also involves a sequence in OEP7 that is similar to the ABS found in APX3.There is no apparent similarity, at the amino acid level, between the sequences of the AKR2A-binding site found in APX3 and OEP7; it appears that what AKR2A recognizes in its ligand proteins is the structural feature: single transmembrane domain followed by one or a few positively charged amino acid residues. Therefore, these AKR2A-binding sequences should all be designated as ABS, and it was predicted that any protein with an ABS could be AKR2A''s interacting protein. Five such proteins, APX5, TOC34, TOM20, cytochrome b5 (CB5) and cytochrome b₅ reductase (CB5R) were tested, and indeed all five proteins interacted with AKR2A in the yeast two-hybrid system.¹ More importantly, the interaction sites of these proteins are their ABS in every case tested.¹ Based on these discoveries, it is proposed that AKR2A is a molecular chaperone for this group of ABS-containing proteins.Among the seven AKR2A-interacting proteins that were characterized, the ABS is found at C-terminal end of four proteins (APX3, APX5, CB5 and TOM20), near N-terminal end of two proteins (OEP7 and CB5R), and near C-terminal end of one protein (TOC34), suggesting that the position of ABS in these membrane proteins does not affect its interaction with AKR2A. Furthermore, in all cases, AKR2A binds to its ligand proteins that contain only one ABS. AKR2A does not appear to bind to proteins that contain multiple transmembrane domains such as PMP22,^1,6 even though these transmembrane domains are followed by a few positively charged amino acid residues.APX3 and APX5 are peroxisomal membrane-bound, OEP7 and TOC34 are chloroplast outer envelope proteins, TOM20 is a mitochondrion outer membrane protein and CB5 and CB5R are microsomal membrane (ER-membrane) proteins. Therefore, AKR2A is clearly not responsible for targeting these proteins to their specific membranes; instead AKR2A serves as a molecular chaperone to prevent these proteins from forming aggregates through their hydrophobic domain in ABS after translation (Fig. 2). Perhaps, AKR2A''s binding to the ABS of these membrane proteins also keeps these proteins in insertion competent state before they are sent to their specific destinations. It is clear that other factors, such as organellar membrane-specific receptors, must be required for sending these proteins to their specific membranes (Fig. 2).Open in a separate windowFigure 2Model on how AKR2A chaperones its ligand proteins. (1) AKR2A binds to ABS of a nascent protein that is being synthesized from a free ribosome. (2) AKR2A keeps its ligand protein (L) in the cytoplasm. (3) With the help of membrane-specific receptors, AKR2A''s ligand proteins are sent to their specific membranes.The Arabidopsis proteome was analyzed and it was found that there are at least 500 proteins that contain sequences similar to ABS (http://bio.scu.edu.cn/list.xls). Would these proteins be AKR2A''s ligand proteins? Some of them, if not all, might be, but it will be a challenging task to experimentally test these proteins one by one. A better bioinformatics tool that can provide clues on the mode of action of the protein-protein interactions between AKR2A and its known ligand proteins should help us designing next set of experiments in order to answer the above question in an efficient way.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

Radiation damping compensation of selective pulses in water–protein exchange spectroscopy

The observation of nuclear Overhauser effects (NOEs) between bound water and biological macromolecules such as proteins and nucleic acids can be improved by inverting the water resonance selectively while compensating for radiation damping effects. The efficiency of inversion, the offset profiles, and the appearance of 2D NOE-NOESY spectra can be improved in comparison with earlier methods.     

Detection of very weak side chain–main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins by sensitivity-enhanced NMR spectroscopy

We describe the direct observation of very weak side chain–main chain hydrogen bonding interactions in medium-size ¹³C/¹⁵N-labeled proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the hydrogen acceptor side chain carboxylate carbon ¹³CO₂ of glutamate 54 and the hydrogen donor backbone amide ¹⁵N of methionine 49 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _NCO2 coupling by employing a novel sensitivity-enhanced HNCO-type experiment, CPD-HNCO. The ^3h J _NCO2 coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains.     

Review of NMR and μSR studies in the molecular nanomagnet Mn12-ac

We present a comprehensive review of the NMR and μSR studies performed in the molecular nanomagnet Mn12 a system characterized and widely studied by Prof. Gatteschi’s group in Florence. The proton (¹H, ²D) NMR, the ⁵⁵Mn NMR and the μSR investigations have yielded important information regarding both static and dynamic magnetic properties of the molecule. The magnetic and quadrupole hyperfine interactions have been extracted from NMR data. The spin dynamics at high and intermediate temperature associated with the zero dimensionality and with the spin–phonon coupling has been studied together with the spin dynamics in the quantum tunneling low temperature regime. The local spin configuration in the giant S = 10 total spin ground state has been determined via ⁵⁵Mn NMR in zero magnetic field and with fields parallel and perpendicular to the anisotropy axis. Finally a novel method is described to monitor the relaxation of the magnetization from the time evolution of the NMR spectrum.     

NMR detection of side chain–side chain hydrogen bonding interactions in 13C/15N-labeled proteins

We describe the direct observation of side chain–side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen ¹⁵N of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon ¹³CO₂ of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _{N CO2} coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The ^3h J _{N CO2} coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.     

A 4D TROSY-based pulse scheme for correlating 1HNi,15Ni,13Cαi,13C′i−1 chemical shifts in high molecular weight, 15N,13C, 2H labeled proteins

A 4D TROSY-based triple resonance experiment, 4D-HNCO_i–1CA_i, is presented which correlates intra-residue ¹HN, ¹⁵N, ¹³ C chemical shifts with the carbonyl (¹³C) shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (¹HN,¹⁵N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCO_i–1CA_i often allows resolution of the ambiguity by linking the ¹³C and ¹³C spins surrounding the (¹HN,¹⁵N) pair. The experiment is demonstrated on a sample of ¹⁵N, ¹³C, ² H labeled maltose binding protein in complex with -cyclodextrin that tumbles with a correlation time of 46 ns.     

Design, conformational studies and analysis of structure–function relationships of PTH (1–11) analogues: the essential role of Val in position 2

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it elicits all the biological responses characteristic of the native intact PTH. Recent studies reported potent helical analogues of the PTH (1-11) with helicity-enhancing substitutions. This work describes the synthesis, biological activity, and conformational studies of analogues obtained from the most active non-natural PTH (1-11) peptide H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2; specifically, the replacement of Val in position 2 with D-Val, L-(αMe)-Val and N-isopropyl-Gly was studied. The synthesized analogues were characterized functionally by in-cell assays and their structures were determined by CD and NMR spectroscopy. To clarify the relationship between the structure and activity, the structural data were used to generate a pharmacophoric model, obtained overlapping all the analogues. This model underlines the fundamental functional role of the side chain of Val2 and, at the same time, reveals that the introduction of conformationally constrained Cα-tetrasubstituted α-amino acids in the peptides increases their helical content, but does not necessarily ensure significant biological activity.     

Molecular dynamics simulation studies of induced fit and conformational capture in U1A–RNA binding: Do molecular substates code for specificity?

Molecular dynamics (MD) simulations on stem loop 2 of U1 small nuclear RNA and a construct of the U1A protein were carried out to obtain predictions of the structures for the unbound forms in solution and to elucidate dynamical aspects of induced fit upon binding. A crystal structure of the complex between the U1A protein and stem loop 2 RNA and an NMR structure for the uncomplexed form of the U1A protein are available from Oubridge et al. (Nature, 1994, Vol. 372, pp. 432-438) and Avis et al. (Journal of Molecular Biology, 1996, Vol. 257, pp. 398-411), respectively. As a consequence, U1A-RNA binding is a particularly attractive case for investigations of induced fit in protein-nucleic acid complexation. When combined with the available structural data, the results from simulations indicate that structural adaptation of U1A protein and RNA define distinct mechanisms for induced fit. For the protein, the calculations indicate that induced fit upon binding involves a non-native thermodynamic substate in which the structure is preorganized for binding. In contrast, induced fit of the RNA involves a distortion of the native structure in solution to an unstable form. However, the RNA solution structures predicted from simulation show evidence that structures in which groups of bases are favorably oriented for binding the U1A protein are thermally accessible. These results, which quantify with computational modeling recent proposals on induced fit and conformational capture by Leuillot and Varani (Biochemistry, 2001, Vol. 40, pp. 7947-7956) and by Williamson (Nature Structural Biology, 2000, Vol. 7, pp. 834-837) suggest an important role for intrinsic molecular architecture and substates other than the native form in the specificity of protein-RNA interactions.     

Site-specific protein methyl deuterium quadrupolar patterns by proton-detected 3D 2H–13C–1H MAS NMR spectroscopy

Journal of Biomolecular NMR - Determination of protein structure and dynamics is key to understand the mechanism of protein action. Perdeuterated proteins have been used to obtain high...