CO Chemical Shifts Research Articles

Asymmetry-enhanced 59Co NMR thermometry in Co(iii) complexes.

Design strategies for molecular thermometers by magnetic resonance are essential for enabling new noninvasive means of temperature mapping for disease diagnoses and treatments. Herein we demonstrate a new design strategy for thermometry based on chemical control of the vibrational partition function. To do so, we performed variable-temperature 59Co NMR investigations of four air-stable Co(iii) complexes: Co(accp)3 (1), Co(bzac)3 (2), Co(tBu2-acac)3 (3), and Co(acac)3 (4) (accp = 2-acetylcyclopentanonate; bzac = benzoylacetonate; tBu2-acac = 2,2,6,6-tetramethyl-3,5-heptanedionate and acac = acetylacetonate). We discovered 59Co chemical shift temperature sensitivity (Δδ/ΔT) values of 3.50(2), 3.39(3), 1.63(3), and 2.83(1) ppm °C-1 for 1-4, respectively, at 100 mM concentration. The values observed for 1 and 2 are new records for sensitivity for low-spin Co(iii) complexes. We propose that the observed heightened sensitivities for 1 and 2 are intimately tied to the asymmetry of the accp and bzac ligands versus the acac and tBu2-acac ligands, which enables a larger number of low-energy Raman-active vibrational modes to contribute to the observed Δδ/ΔT values.

Molecular Magnetic Materials Based on {CoIII (Tp*)(CN)3 }- Cyanidometallate: Combined Magnetic, Structural and 59 Co NMR Study.

The cyanidocobaltate of formula fac‐PPh4[CoIII(Me2Tp)(CN)3] ⋅ CH3CN (1) has been used as a metalloligand to prepare polynuclear magnetic complexes (Me2Tp=hydrotris(3,5‐dimethylpyrazol‐1‐yl)borate). The association of 1 with in situ prepared [FeII(bik)2(MeCN)2](OTf)2 (bik=bis(1‐methylimidazol‐2‐yl)ketone) leads to a molecular square of formula {[CoIII{(Me2Tp)}(CN)3]2[FeII(bik)2]2}(OTf)2 ⋅ 4MeCN ⋅ 2H2O (2), whereas the self‐assembly of 1 with preformed cluster [CoII 2(OH2)(piv)4(Hpiv)4] in MeCN leads to the two‐dimensional network of formula {[CoII 2(piv)3]2[CoIII(Me2Tp)(CN)3]2 ⋅ 2CH3CN}∞ (3). These compounds were structurally characterized via single crystal X‐ray analysis and their spectroscopic (FTIR, UV‐Vis and 59Co NMR) properties and magnetic behaviours were also investigated. Bulk magnetic susceptibility measurements reveal that 1 is diamagnetic and 3 is paramagnetic throughout the explored temperature range, whereas 2 exhibits sharp spin transition centered at ca. 292 K. Compound 2 also exhibits photomagnetic effects at low temperature, selective light irradiations allowing to promote reversibly and repeatedly low‐spin⇔high‐spin conversion. Besides, the diamagnetic nature of the Co(III) building block allows us studying these compounds by means of 59Co NMR spectroscopy. Herein, a 59Co chemical shift has been used as a magnetic probe to corroborate experimental magnetic data obtained from bulk magnetic susceptibility measurements. An influence of the magnetic state of the neighbouring atoms is observed on the 59Co NMR signals. Moreover, for the very first time, 59Co NMR technique has been successfully introduced to investigate molecular materials with distinct magnetic properties.

Isotopomeric Elucidation of the Mechanism of Temperature Sensitivity in 59Co NMR Molecular Thermometers.

Understanding the mechanisms governing temperature-dependent magnetic resonance properties is essential for enabling thermometry via magnetic resonance imaging. Herein we harness a new molecular design strategy for thermometry─that of effective mass engineering via deuteration in the first coordination shell─to reveal the mechanistic origin of 59Co chemical shift thermometry. Exposure of [Co(en)3]3+ (1; en = ethylenediamine) and [Co(diNOsar)]3+ (2; diNOsar = dinitrosarcophagine) to mixtures of H2O and D2O produces distributions of [Co(en)3]3+-dn (n = 0-12) and [Co(diNOsar)]3+-dn (n = 0-6) isotopomers all resolvable by 59Co NMR. Variable-temperature 59Co NMR analyses reveal a temperature dependence of the 59Co chemical shift, Δδ/ΔT, on deuteration of the N-donor atoms. For 1, deuteration amplifies Δδ/ΔT by 0.07 ppm/°C. Increasing degrees of deuteration yield an opposing influence on 2, diminishing Δδ/ΔT by -0.07 ppm/°C. Solution-phase Raman spectroscopy in the low-frequency 200-600 cm-1 regime reveals a red shift of Raman-active Co-N6 vibrational modes by deuteration. Analysis of the normal vibrational modes shows that Raman modes produce the largest variation in 59Co δ. Finally, partition function analysis of the Raman-active modes shows that increased populations of Raman modes predict greater Δδ/ΔT, representing new experimental insight into the thermometry mechanism.

Cobalt(III) Metallacryptates and Their Guest Cation-Exchange in Solution Monitored by 59Co NMR.

One-pot reactions of the catechol-scaffolding aroylbis(N,N-diethylthiourea) H2Lcat with mixtures of CoCl2 and MCl (M+ = Cs+, Rb+, K+, Tl+, or NH4+) or with a Co(NO3)2/TlNO3 mixture lead to the self-assembly of a series of cationic Co(III) metallacryptates of the general formula [M ⊂ {Co2(Lcat)3}]+ (M+ = Cs+, Rb+, K+, Tl+, or NH4+). Crystalline PF6- salts were obtained after workup with (n-Bu4N)(PF6), and the single-crystal structures of all five metallacryptates have been determined. Depending on the nature of the guest cations, the directional interactions between guest cations and the metallacryptand {Co2(Lcat)3} are either weak coordination contacts or hydrogen bonds. The bonding mode and the size of the guest ions slightly influence the molecular skeleton of the host molecule. These small structural variations also exist in solution and could be detected by means of 59Co NMR spectroscopy, which is shown to be a unique tool for an easy characterization of such compounds. 59Co NMR chemical shifts are extraordinarily sensitive to the guest cation in the metallacryptates, and time-arrayed 59Co NMR experiments show that cation-exchange processes in biphasic organic/aqueous systems can be studied in detail. This leads to insights into the relative rates of cation exchange, as well as the relative conditional distribution coefficients of such Co(III) metallacryptates between the aqueous and organic phases. Thus, the extent and the relative rate of the NH4+ ion exchange in [NH4 ⊂ {Co2(Lcat)3}](PF6) by Cs+ and K+ ions across the organic/aqueous phase boundary at room temperature have been studied by in situ 59Co NMR experiments. Preliminary 59Co NMR experiments show that the K+ ion in [K ⊂ {Co2(Lcat)3}](PF6) can be removed by its competitive complexation with the highly potassium-selective [2.2.2]cryptand, to give a transient 59Co NMR signal of the relatively unstable "empty" {Co2(Lcat)3} complex, which slowly decomposes in solution.

13C NMR substituent-induced chemical shifts in 4-(substituted phenyl)-3-phenyl-1,2,4-oxadiazol-5(4H)-ones (thiones)

In the present, study mostly novel ten 4-(substituted phenyl)-3-phenyl-1,2,4-oxadiazol-5(4H)-ones and ten 4-(substituted phenyl)-3-phenyl-1,2,4-oxadiazol-5(4H)-thiones were synthesized. These oxadiazole derivatives were characterized by IR, 1H NMR, 13C NMR and elemental analyses. Their 13C NMR spectra were measured in Deuterochloroform (CDCl3). The correlation analysis for the substituent-induced chemical shift (SCS) with Hammett substituent constants (σ), Brown Okamoto substituent constants (σ+, σ−), inductive substituent constants (σI) and different of resonance substituent constants (σR, σRo) were performed using SSP (single substituent parameter), DSP (dual substituent parameter) and DSP-NLR (dual substituent parameter-non-linear resonance) methods, as well as single and multiple regression analysis. Negative ρ values were found for all correlations (reverse substituent effect). The results of all statistical analyses, 13C NMR chemical shift of CN, CO and CS carbon of oxadiazole rings have shown satisfactory correlation.

A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins.

Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue-HN, N, CA and CO chemical shifts-from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i - 1) amide (1)H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135-147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.

Parallel acquisition of multi-dimensional spectra in protein NMR

We introduce the use of multiple receivers applied in parallel for simultaneously recording multi-dimensional data sets of proteins in a single experiment. The utility of the approach is established through the introduction of the 2D (15)N,(1)H(N)||(13)CO HSQC experiment in which a pair of two-dimensional (15)N,(1)H(N) and (15)N,(13)CO spectra are recorded. The methodology is further extended to higher dimensionality via the 3D (1)H(N)||(13)CO HNCA in which a pair of data sets recording (13)C(α),(15)N,(1)H(N) and (13)C(α),(15)N,(13)CO chemical shifts are acquired. With the anticipated increases in probe sensitivity it is expected that multiple receiver experiments will become an important approach for efficient recording of NMR data.

Backbone assignment of perdeuterated proteins using long-range H/C-dipolar transfers

For micro-crystalline proteins, solid-state nuclear magnetic resonance spectroscopy of perdeuterated samples can provide spectra of unprecedented quality. Apart from allowing to detect sparsely introduced protons and thereby increasing the effective resolution for a series of sophisticated techniques, deuteration can provide extraordinary coherence lifetimes--obtainable for all involved nuclei virtually without decoupling and enabling the use of scalar magnetization transfers. Unfortunately, for fibrillar or membrane-embedded proteins, significantly shorter transverse relaxation times have been encountered as compared to micro-crystalline proteins despite an identical sample preparation, calling for alternative strategies for resonance assignment. In this work we propose an approach towards sequential assignment of perdeuterated proteins based on long-range (1)H/(13)C Cross Polarization transfers. This strategy gives rise to H/N-separated correlations involving C(α), C(β), and CO chemical shifts of both, intra- and interresidual contacts, and thus connecting adjacent residues independent of transverse relaxation times.

Protegrin‐1 orientation and physicochemical properties in membrane bilayers studied by potential of mean force calculations

Protegrin-1 (PG-1) belongs to the family of antimicrobial peptides. It interacts specifically with the membrane of a pathogen and kills the pathogen by releasing its cellular contents. To fully understand the energetics governing the orientation of PG-1 in different membrane environments and its effects on the physicochemical properties of the peptide and membrane bilayers, we have performed the potential of mean force (PMF) calculations as a function of its tilt angle at four distinct rotation angles in explicit membranes composed of either DLPC (1,2-dilauroylphosphatidylcholine) or POPC (1-palmitoyl-2-oleoylphosphatidylcholine) lipid molecules. The resulting PMFs in explicit lipid bilayers were then used to search for the optimal hydrophobic thickness of the EEF1/IMM1 implicit membrane model in which a two-dimensional PMF in the tilt and rotation space was calculated. The PMFs in explicit membrane systems clearly reveal that the energetically favorable tilt angle is affected by both the membrane hydrophobic thickness and the PG-1 rotation angle. Local thinning of the membrane around PG-1 is observed upon PG-1 tilting. The thinning is caused by both hydrophobic mismatch and arginine-lipid head group interactions. The two-dimensional PMF in the implicit membrane is in good accordance with those from the explicit membrane simulations. The ensemble-averaged Val16 (15)N and (13)CO chemical shifts weighted by the two-dimensional PMF agree fairly well with the experimental values, suggesting the importance of peptide dynamics in calculating such ensemble properties for direct comparison with experimental observables.

Protegrin-1 Orientation in Membrane Bilayers: Insights from Potential of Mean Force Calculations as A Function of Its Tilt and Rotation Angles

Protegrin-1 (PG-1) belongs to the family of antimicrobial peptides. It interacts specifically with the membrane of a pathogen and kills the pathogen by releasing its cellular contents. To fully understand the energetics governing the orientation of PG-1 in different membrane environments and its effects on the physicochemical properties of the peptide, we have calculated the potentials of mean force (PMF) of PG-1 as a function of its tilt angle in explicit membrane bilayers composed of either DLPC (1,2-dilauroylphosphatidylcholine) or POPC (1-palmitoyl-2-oleoylphosphatidylcholine) lipid molecules. The resulting PMFs in explicit lipid bilayers were then used to search for the optimal hydrophobic thickness of the implicit membrane, in which a two-dimensional PMF in the tilt and rotation space was calculated. The calculated PMFs in explicit membrane systems clearly reveal that the energetically favorable tilt angle is affected by both the membrane hydrophobic thickness and the PG-1 rotation angle. Local thinning of the membrane around PG-1 is observed upon PG-1 tilting. The thinning effect is caused by different arginines in regard to the rotation orientation of the peptide. The two-dimensional PMF calculated in implicit membrane at specified rotation angles is in good accordance with those from the explicit membrane simulations. The ensemble-averaged Val16 15N and 13CO chemical shifts calculated from the two-dimensional free energy distribution agree fairly well with the experimental values, suggesting the accuracy and reliability of the application of PMFs to understand important physicochemical properties of membrane peptides/proteins.

Comparative Molecular Dynamics Simulation Studies of Protegrin-1 Monomer and Dimer in Two Different Lipid Bilayers

Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated 15N and 13CO chemical shifts of Val16 in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions.

Native like structure in the unfolded state of the villin headpiece helical subdomain, an ultrafast folding protein

The villin headpiece subdomain, HP36, is the smallest naturally occurring protein that folds cooperatively. Its small size, rapid folding, and simple three-helix topology have made it an extremely popular system for computational studies of protein folding. The role of unfolded state structure in rapid folding is an area of active investigation, but relatively little is known about the properties of unfolded states under native conditions. A peptide fragment, HP21, which contains the first and second helices of HP36 has been shown to be a good model for structure in the unfolded state of the intact domain but a detailed description of the conformational propensities of HP21 is lacking and the balance between native and nonnative interactions is not known. A series of three-dimensional NMR experiments were performed on (13)C, (15)N-labeled HP21 to investigate in detail its conformational propensities. Analysis of (13)C(alpha), (13)C(beta), (13)CO chemical shifts, Deltadelta(13)C(alpha) - Deltadelta(13)C(beta) secondary shifts, the secondary structure propensity scores, NOEs, (15)N R(2) values and comparison of experimental chemical shifts with those of HP36 and with chemical shifts calculated using the SHIFTS and SHIFTX programs all indicate that there is significant native like structure in the HP21 ensemble, and thus by implication in the unfolded state of HP36.

Co(III) complexes of the type [(L)Co(O 2CO)] + (L = tripodal tetraamine ligand): Synthesis, structure, DFT calculations and 59Co NMR

The syntheses and characterisation of the Co(III) complexes [(L)Co(O 2CO)]ClO 4 (L = a tripodal tetraamine ligand = baep, abap, uns-penp, dppa, trpn) are reported. Geometric isomers are possible for all but the trpn complex, owing to the non-equivalence of the three arms on the tripodal ligand, and both NMR and X-ray crystallography are used to identify the single isomer formed. X-ray crystal structures of the complexes [(L)Co(O 2CO)]ClO 4 · xH 2O (L = baep, x = 0.5; L = abap, x = 0; L = uns-penp, x = 1; L = dppa, x = 0; L = trpn, x = 1) are reported; little variation is observed in the geometry of the carbonate chelate ring while significant lengthening of bonds and expansion of angles involving the cobalt ion occurs as the number of six-membered chelate rings in the complex cations increases. 59Co NMR chemical shift data for the complexes show the expected linear relationship between λ max, the wavelength of the lowest energy d– d transition, and γ, the magnetogyric ratio of the 59Co nucleus. An excellent correlation between Δ, the d orbital splitting parameter, and δ( 59Co) also exists for these complexes. Rate data for the acid hydrolysis of [(L)Co(O 2CO)] + (L = uns-penp, dppa) in 1.0 M HClO 4 differ by two orders of magnitude, and this is attributed to the differing steric accessibility of the endo O atoms in each complex. DFT calculations on the complexes reproduce the isomeric preferences, UV–Vis and 59Co NMR spectroscopic data well, provided that solvent effects are included.

Native Conformation at Specific Residues in Inclusion Body Protein in Whole Cells Detected with Solid-State Nuclear Magnetic Resonance

Recombinant protein expression is often plagued by the production of inclusion bodies which are insoluble aggregates of the expressed protein. Although little is known about the structures of inclusion body protein, one model is amyloid beta sheet structure. Residue-specific conformation of two recombinant proteins in inclusion bodies was probed by solid-state nuclear magnetic resonance. One protein was a 193-residue membrane protein and the other protein was a 92-residue soluble protein. The native conformations were predominantly helical. The 13CO chemical shifts of individual residues in the proteins were measured and correlated with local conformation. Three types of samples were studied: (1) purified protein which had native structure; (2) whole unlysed hydrated cells; and (3) the hydrated pellet formed from the insoluble material in the cell lysate. The 13CO signals from the latter two types of samples were predominantly from inclusion body protein. All measured 13CO chemical shifts in all samples correlated with helical rather than beta strand conformation. The data therefore suggest that much of the native fold is retained for these two proteins in hydrated inclusion bodies.View Large Image | View Hi-Res Image | Download PowerPoint Slide

HIV Fusion Peptide and Its Cross-Linked Oligomers: Efficient Syntheses, Significance of the Trimer in Fusion Activity, Correlation of β Strand Conformation with Membrane Cholesterol, and Proximity to Lipid Headgroups

For enveloped viruses such as HIV, an approximately 20-residue N-terminal fusion peptide domain in the envelope protein binds to target cell membranes and plays a key role in fusion between the viral and cellular membranes during infection. The chemically synthesized HIV fusion peptide (HFP) catalyzes fusion between membrane vesicles and is a useful model system for understanding some aspects of HIV fusion. Previous studies have shown a common trimeric state for the envelope protein from several different viruses, including HIV, and in this study, practical high-yield syntheses are reported for HFP monomer (HFPmn) and chemically cross-linked HFP dimer (HFPdm), trimer (HFPtr), and tetramer (HFPte). The vesicle fusion rates per strand were ordered as follows: HFPmn < HFPdm < HFPtr approximately HFPte. This suggested that HFPtr is the smallest catalytically efficient oligomer. Solid-state NMR measurements of (13)CO chemical shifts were carried out in constructs labeled at either Ala-6 or Ala-15. For all constructs associated with cholesterol-containing membranes, the chemical shifts of both residues correlated with beta strand conformation while association with membranes without cholesterol resulted in a mixture of helical and beta strand conformations. The dependence of fusion rate on oligomer size is independent of membrane cholesterol content, so one interpretation of the data is fusion activity of both helical and beta strand conformations. Membrane location may be a determinant of fusion activity, and for all constructs in both conformations, a large fraction of the Ala-15 (13)CO groups were 5-6 A from the (31)P atoms in the lipid headgroups, while the Ala-6 (13)CO groups were more distant.

High‐resolution NMR characterization of a spider‐silk mimetic composed of 15 tandem repeats and a CRGD motif

Multidimensional solution NMR spectroscopic techniques have been used to obtain atomic level information about a recombinant spider silk construct in hexafluoro-isopropanol (HFIP). The synthetic 49 kDa silk-like protein mimics authentic silk from Nephila clavipes, with the inclusion of an extracellular matrix recognition motif. 2D (1)H-(15)N HSQC NMR spectroscopy reveals 33 cross peaks, which were assigned to amino acid residues in the semicrystalline repeat units. Signals from the amorphous segments in the primary sequence were weak and broad, suggesting that this region is highly dynamic and undergoing conformational exchange. An analysis of the deviations of the (13)C(alpha), (13)C(beta), and (13)CO chemical shifts relative to the expected random coil values reveals two highly alpha-helical regions from amino acid 12-19 and 26-32, which comprise the polyalanine track and a GGLGSQ sequence. This finding is further supported by phi-value analysis and sequential and medium-range NOE interactions. Pulsed field gradient NMR measurements indicate that the topology of the silk mimetic in HFIP is nonglobular. Moreover, the 3D (15)N-NOESY HSQC spectrum exhibits few long-range NOEs. Similar spectral features have been observed for repeat modules in other polypeptides and are characteristic of an elongated conformation. The results provide a residue-specific description of a silk sequence in nonaqueous solution and may be insightful for understanding the fold and topology of highly concentrated, stable silk before spinning. Additionally, the insights obtained may find application in future design and large-scale production and storage of synthetic silks in organic solvents.

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.

Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy.

Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)-ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of (15)N, (1)HN, (13)C(alpha), and (13)CO chemical shifts, along with (1)HN-(15)N, (1)H(alpha)-(13)C(alpha), and (1)HN-(13)CO residual dipolar couplings and (13)CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with (1)HN-(15)N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.

Structural Analysis of Transmembrane Halobacterial Tranceducer pHtrII by Multi-Dimensional High-Resolution Solid-State NMR

°Transmembrane protein pHtrII is a transducer which binds to phoborhodopsin. The light excitation of phoborhodopsin is transmitted into the cytoplasm through pHtrII to promote negative photoaxis. We studied uniformly 13 C, 15 N labeled 159residues pHtrII by high-resolution solid-state NMR. The pHtrII (1-159) was econstructed into the deuterated DMPC membranes. High-resolution solid-state 2D NMR was measured under magic-angle spinning at the 1 H resonance frequencies of 500, 600 and 700 MHz . At first, we have assigned 13 C signals of pHtrII (1-159) by using 13 C- 13 C spin diffusion under DARR at -40 o C.Intraresidue correlations across 1-2 bonds were observed at short mixing time. At a long mixing time of 200 ms, intra-residue C α correlations across 3-bonds were obtained. Most amino acids except Tyr and Pro were assigned. The DARR spectra were observed for immobile region of pHtrII. All 13 C signals could not be observed in these dipolar-coupling-based cross-polarization methods because of large-amplitude molecular motions at room temperature. We performed Jcoupling-based HC-INEPT and HCC-TOCSY experiments in order to obtain signals of mobile protein segments. Mobile protein segments make up about 70% of pHtrII (1-159). Many signals in mobile regions were observed in HC and HCC 2D spectra. 13 C and 1 H signals were assigned to amino acid groups were successful in mobile regions. The C α and CO chemical shifts indicate that pHtrII (1-159) mainly forms α -helix structure in lipid bilayer environment.

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A 600 ∼ 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the 13CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy.