In-cell solid-state NMR as a tool to study proteins in large complexes.

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.

Dynamic nuclear polarization-enhanced solid-state NMR of a 13C-labeled signal peptide bound to lipid-reconstituted Sec translocon.

Dynamic nuclear polarization (DNP) has made it possible to record 2D double-quantum-filtered (DQF) solid-state NMR (ssNMR) spectra of a signal peptide bound to a lipid-reconstituted SecYEG translocon complex. The small quantity of peptide in the sample (~40 nmol) normally prohibits multidimensional ssNMR experiments. Such small amounts are not the exception, because for samples involving membrane proteins, most of the limited sample space is occupied by lipids. As a consequence, a conventional 2D DQF ssNMR spectrum with the sample used here would require many weeks if not months of measurement time. With the help of DNP, however, we were able to acquire such a 2D spectrum within 20 h. This development opens up new possibilities for membrane protein studies, particularly in the exploitation of high-resolution spectroscopy and the assignment of individual amino acid signals, in this case for a signal peptide bound to the translocon complex.

Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR.

We describe a simple yet highly effective optimization strategy for SPINAL-64 ¹H decoupling conditions for magic-angle spinning solid-state NMR. With adjustment of the phase angles in a coupled manner, the optimal conditions resulting from three parameter optimizations can be determined with adjustment of a single phase. Notably, echo T2 relaxation times for ¹³C and ¹5N show significant enhancement (up to 64%), relative to the previous described SPINAL-64 conditions, under the moderate ¹H decoupling levels (60-100 kHz) and MAS rate (13.3 kHz) commonly employed for high-resolution SSNMR spectroscopy of proteins. Additionally, we also investigated the effect at higher spinning rate (33.3 kHz) and compared the results with other ¹H decoupling schemes (TPPM, XiX), as well as SPINAL-64 with the originally reported optimal values.

Hyperforin induces Ca(2+)-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A(2) activation through select phospholipid interactions.

Here, we investigated the modulation of cytosolic phospholipase A(2) (cPLA(2))-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10microM) versus unstimulated cells, which was blocked by cPLA(2)alpha-inhibition, and induced translocation of cPLA(2) to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca(2+) by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA(2) at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA(2) phosphorylation. However, neither AA release nor translocation of cPLA(2) was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA(2) in the presence of Ca(2+). However, when Ca(2+) was omitted, hyperforin caused a prominent increase in cPLA(2) activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional (1)H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA(2) activation by enabling its access towards select lipid membranes independent of Ca(2+) ions. Such Ca(2+)- and phosphorylation-independent mechanism of cPLA(2) activation may apply also to other membrane-interfering molecules.

Higher sensitivity through selective (13)C excitation in solid-state NMR spectroscopy.

A notable drawback of NMR spectroscopy is its inherently low sensitivity: 95% of the measuring time consists solely of idle delays during which nuclei regain their Boltzmann equilibrium. Here, a strategy for solid-state (13)C NMR experiments is presented that allows the user to acquire spectra in time periods that are notably shorter than previously necessary. Experiments that are band-selective in nature may utilize the cooling potential of unperturbed nuclei to lower the spin temperature of their excited neighbors. As we demonstrate, it becomes possible to replace the recycle delay in a series of scans by a time period during which proton-driven spin diffusion causes relaxation enhancement by a lower spin temperature of adjacent spins (RELOAD). Typically, a duration of approximately 200 ms suffices for this step, and for 1D (13)C NMR experiments, it is shown that the omission of recycle delays (typically of 2 s length) reduces the measuring time substantially. RELOAD is applied to 2D homonuclear (13)C NMR experiments, and it is demonstrated that for experiments in which correlations between (13)C backbone atoms are detected, the measurement time is reduced by a factor of 10 through a time-saving combination of a smaller number of increments in the indirect dimension and RELOAD.

RNA phosphodiester backbone dynamics of a perdeuterated cUUCGg tetraloop RNA from phosphorus-31 NMR relaxation analysis.

We have analyzed the relaxation properties of all (31)P nuclei in an RNA cUUCGg tetraloop model hairpin at proton magnetic field strengths of 300, 600 and 900 MHz in solution. Significant H, P dipolar contributions to R (1) and R (2) relaxation are observed in a protonated RNA sample at 600 MHz. These contributions can be suppressed using a perdeuterated RNA sample. In order to interpret the (31)P relaxation data (R (1), R (2)), we measured the (31)P chemical shift anisotropy (CSA) by solid-state NMR spectroscopy under various salt and hydration conditions. A value of 178.5 ppm for the (31)P CSA in the static state (S (2) = 1) could be determined. In order to obtain information about fast time scale dynamics we performed a modelfree analysis on the basis of our relaxation data. The results show that subnanosecond dynamics detected around the phosphodiester backbone are more pronounced than the dynamics detected for the ribofuranosyl and nucleobase moieties of the individual nucleotides (Duchardt and Schwalbe, J Biomol NMR 32:295-308, 2005; Ferner et al., Nucleic Acids Res 36:1928-1940, 2008). Furthermore, the dynamics of the individual phosphate groups seem to be correlated to the 5' neighbouring nucleobases.

Cellular membranes function as a storage compartment for celecoxib.

Celecoxib is a selective cyclooxygenase-2-(COX-2)-inhibitor used to treat inflammation and pain and prevents colorectal cancer in patients at high doses by affecting several non-COX-2 proteins. However, celecoxib concentrations appropriate to inhibit proliferation or to induce apoptosis in cell culture (up to 100 microM) clearly exceed those in human plasma (up to 10 microM). Therefore, we speculated that celecoxib might accumulate in human cells, which may facilitate the drug's interaction with non-COX-2 proteins. Determination of intracellular celecoxib concentrations by liquid chromatography tandem mass spectrometry gave five- to tenfold higher levels as compared to other coxibs (etoricoxib, valdecoxib, lumiracoxib, and rofecoxib) in different tumor cell types, including human HCA-7 and HCT-116 colon carcinoma cells, BL-41 B lymphocytes, Mono Mac 6 monocytes, and in mouse NIH-3T3 non-tumor fibroblasts. This intracellular accumulation of celecoxib was due to an integration of the drug into cellular phospholipid membranes as demonstrated by nuclear Overhauser spectroscopy/nuclear magnetic resonance. Consequently, celecoxib disturbed the plasma membrane integrity of HCT-116 cells and displayed an increased COX-2-inhibitory potency in HCA-7 cells. The use of other coxibs demonstrated that intracellular accumulation is peculiar of celecoxib. Accumulation of celecoxib in human cells may provide a novel molecular basis for the ability of the drug to interact with non-COX-2 targets in vivo despite comparatively low plasma concentrations.

A practical synthesis of the 16C/15N-labelled tripeptide N-formyl-Met-Leu-Phe, useful as a reference in solid-state NMR spectroscopy.

A mild synthetic method for N-formyl-Met-Leu-Phe-OH (1) is described. After Fmoc solid phase peptide synthesis, on-bead formylation and HPLC purification, more than 30 mg of the fully (13)C/(15)N-labelled tripeptide 1 could be isolated in a typical batch. This peptide can be easily crystallised and is therefore well suited as a standard sample for setting up solid-state NMR experiments.

Theoretical study of the human bradykinin-bradykinin B2 receptor complex.

The interaction of bradykinin (BK) with the bradykinin B2 receptor (B2R) was analyzed by using molecular modeling (MM) and molecular dynamics (MD) simulations. A homology model for B2R has been generated and the recently determined receptor-bound solid-state NMR spectroscopic structure of BK (Lopez et al., Angew. Chem. 2008, 120, 1692-1695; Angew. Chem. Int. Ed. 2008, 47, 1668-1671) has been modeled into the binding pocket of the receptor to probe the putative ligand-receptor interface. The experimental hormone structure fitted well into the binding pocket of the receptor model and remained stable during the MD simulation. We propose a parallel orientation of the side chains for Arg1 and Arg9 in BK that is bound to B2R. The MD simulation study also allows the conformational changes that lead to the activated form of B2R to be analyzed. The hydrogen bond between N140 (3.35) and W283 (6.48) is the key interaction that keeps the receptor in its inactive form. This hydrogen bond is broken during the MD simulation due to rotation of transmembrane helix 3 (TM3) and is replaced by a new hydrogen bond between W283 (6.48) and N324 (7.45). We propose that this interaction is specific for the activated form of the bradykinin B2 receptor. Additionally, we compared and discussed our putative model in the context of the structural model of the partially activated rhodopsin (Rh*) and with the known biochemical and structural data.

Double quantum filtering homonuclear MAS NMR correlation spectra: a tool for membrane protein studies.

13C homonuclear correlation spectra based on proton driven spin diffusion (PDSD) are becoming increasingly important for obtaining distance constraints from multiply labeled biomolecules by MAS NMR. One particular challenging situation arises when such constraints are to be obtained from spectra with a large natural abundance signal background which causes detrimental diagonal peak intensities. They obscure cross peaks, and furthermore impede the calculation of a buildup rates matrix which may be used to derive distance constraints, as carried out in "NMR crystallography". Here, we combine double quantum (DQ) filtering with 13C-13C dipolar assisted rotational resonance (DARR) experiments to yield correlation spectra free of natural abundance contributions. Two experimental schemes, using DQ filtering prior to evolution (DOPE), and after mixing (DOAM), have been evaluated. Diagonal peak intensities along the spectrum diagonal are removed completely, and crosspeaks close to the diagonal are easily identifiable. For DOAM spectra with negligible mixing times, it is possible to carry out 'assignment walks' which simplify peak identification substantially. The method is demonstrated on 13C-cys labeled proteorhodopsin, a 27 kDa membrane protein. The magnetization transfer characteristics were studied using buildup curves obtained on uniformly 13C labelled crystalline tripeptide MLF. Our data show that DQ filtered DARR experiments pave the way for obtaining through space constraints for structural studies on ligands, bound to membrane receptors, or on small fragments within large proteins.

Location and orientation of serotonin receptor 1a agonists in model and complex lipid membranes.

Magic angle spinning (MAS) NMR has been used to investigate the location and orientation of five serotonin receptor 1a agonists (serotonin, buspirone, quipazine, 8-OH-DPAT, and LY-163,165) in single component model lipid and brain lipid membranes. The agonist locations are probed by monitoring changes in the lipid proton chemical shifts and by MAS-assisted nuclear Overhauser enhancement spectroscopy, which indicates the orientation of the agonists with respect to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids. In the single component bilayer, the membrane agonists are found predominantly in the top of the hydrophobic chain or in the glycerol region of the membrane. Most of the agonists orient approximately parallel to the membrane plane, with the exception of quipazine, whose piperazine ring is found in the glycerol region, whereas its benzene ring is located within the lipid hydrophobic chain. The location of the agonist in brain lipid membranes is similar to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers; however, many of the agonists appear to locate close to the cholesterol in the membrane in preference to the phospholipids.

Dual transformation of homonuclear solid-state NMR spectra--an option to decrease measuring time.

Long measurement times due to low sensitivity are a prime concern in solid-state NMR and limit the application of multidimensional experiments severely. One possibility to address this problem could be post-experimental suppression of noise and a reduction of the number of increments needed for higher dimensional data sets. This can be achieved by a hybrid approach based on the combination of separately Fourier transformed and covariance processed datasets. The method is applied to synthetic sets as well as to experimental two-dimensional homonuclear solid-state NMR spectra of peptide samples. It is demonstrated that a reduction in experiment time by a factor of 4 can be achieved for the case of a 13C-13C correlation spectrum on the nonapeptide bradykinin.

Probing the molecular dynamics of the ABC multidrug transporter LmrA by deuterium solid-state nuclear magnetic resonance.

The molecular dynamics of the 64 kDa ABC multidrug efflux pump LmrA from Lactococcus lactis within lipid membranes has been investigated by deuterium solid-state NMR. Deuteriomethyl-labeled alanine has been used to probe global protein backbone dynamics. A comparison of static deuterium NMR spectra of full-length LmrA in the resting state and its isolated transmembrane domain revealed a high mobility for the nucleotide binding domains. Their motional freedom is restricted upon ATP binding as seen for LmrA in complex with AMP-PNP, a nonhydrolyzable ATP analogue. LmrA returns to full motional flexibility in the posthydrolysis, vanadate-trapped state. These experiments provide insight into the molecular dynamics of a full-length ABC transporter during the catalytic cycle. Data are discussed in the context of known biochemical data and structural models of ABC transporters.

Separated local field NMR experiments on oriented samples rotating at the magic angle.

Biophysical studies on membrane proteins by solid state NMR (SSNMR) can be carried out directly in a membrane environment. Samples are usually prepared in form of multi-lamellar dispersions for magic angle sample spinning or as aligned multi-layers for orientation dependent NMR experiments without sample rotation. A new development is the application of MAS NMR to aligned samples (MAOSS; Magic Angle Oriented Sample Spinning). In combination with separated local field (SLF) experiments, size and orientation of heteronuclear dipolar couplings may be extracted from two-dimensional experiments which correlate dipolar couplings with isotropic chemical shifts. The orientation of these (1)H-X dipolar couplings can be directly related to the orientation of molecular groups in the sample. Here, we demonstrate the feasibility of these experiments on highly ordered polyethylene fibers which serve as model compound. Based on these data, the experiment is also applied to ordered multi-layers of bacteriorhodopsin (purple membrane) which is used as a model for aligned membrane proteins. We present a detailed analysis of different experimental designs with respect to angular sensitivity and the influence of residual sample disorder ("mosaic spread"). The results of the MAOSS-SLF experiment are discussed within the context of established solid state NMR experiments which are usually performed without sample rotation and we compare the data to orientation information obtained from X-ray diffraction.

Localization of multidrug transporter substrates within model membranes.

Active extrusion of drugs from the cell interior by primary and secondary efflux pumps is an essential mechanism underlying the phenomenon of multidrug resistance. The first discovered and best characterized primary efflux pump found in humans is the ABC transporter P-glycoprotein (PGP), which shows very broad substrate specificity. Many of these molecules are lipophilic, and binding most likely takes place within the membrane. PGP could either translocate them from the inner to the outer leaflet (flippase) or extrude them from the membrane into the extracellular environment (hydrophobic vacuum cleaner). Recognition and binding of such a diverse set of substrates must be associated with a preferred membrane location, determined by molecular properties and lipid interactions. Therefore, a systematic study of the interaction among seven PGP substrates (phenazine, doxorubicin, cephalexin, ampicillin, chloramphenicol, penicillin G, and quercetin) and two modulators (quinidine and nicardipine) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membranes is reported here. The location profile of these molecules across the membrane was determined by (1)H NOESY MAS NMR based on (1)H-(1)H cross-peaks between their aromatic fingerprint region and lipid resonances. Although structurally rather diverse, all tested substances are found to have their highest concentration between the phosphate of the lipid headgroup and the upper segments of the lipid hydrocarbon chains. Our findings are consistent with PGP substrate and modulator binding from the membrane interface region.

A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39).

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled 31P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from 2H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues.

The oxidative folding of the Amaranthus alpha-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by (1)H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.