共查询到20条相似文献,搜索用时 15 毫秒
1.
Vitaly V. Vostrikov Christopher V. Grant Stanley J. Opella Roger E. Koeppe II 《Biophysical journal》2011,101(12):2939
The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of 2H-alanine quadrupolar splittings together with 15N/1H dipolar couplings and 15N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA)6LWLAGA-amide). The results indicate that derivatives of GWALP23 incorporating diverse guest residues adopt a range of apparent tilt angles that span 5°–35° in lipid bilayer membranes. By comparing individual and combined analyses of specifically 2H- or 15N-labeled peptides incorporated in magnetically or mechanically aligned lipid bilayers, we examine the influence of data-set size/identity, and of explicitly modeled dynamics, on the deduced average orientations of the peptides. We conclude that peptides with small apparent tilt values (<∼10°) can be fitted by extensive families of solutions, which can be narrowed by incorporating additional 15N as well as 2H restraints. Conversely, peptides exhibiting larger tilt angles display more narrow distributions of tilt and rotation that can be fitted using smaller sets of experimental constraints or even with 2H or 15N data alone. Importantly, for peptides that tilt significantly more than 10° from the bilayer-normal, the contribution from rigid body dynamics can be approximated by a principal order parameter. 相似文献
2.
De Angelis AA Grant CV Baxter MK McGavin JA Opella SJ Cotten ML 《Biophysical journal》2011,(5):1086-1094
The amphipathic antimicrobial peptide piscidin 1 was studied in magnetically aligned phospholipid bilayers by oriented-sample solid-state NMR spectroscopy. 31P NMR and double-resonance 1H/15N NMR experiments performed between 25°C and 61°C enabled the lipid headgroups as well as the peptide amide sites to be monitored over a range of temperatures. The α-helical peptide dramatically affects the phase behavior and structure of anionic bilayers but not those of zwitterionic bilayers. Piscidin 1 stabilizes anionic bilayers, which remain well aligned up to 61°C when piscidin 1 is on the membrane surface. Two-dimensional separated-local-field experiments show that the tilt angle of the peptide is 80 ± 5°, in agreement with previous results on mechanically aligned bilayers. The peptide undergoes fast rotational diffusion about the bilayer normal under these conditions, and these studies demonstrate that magnetically aligned bilayers are well suited for structural studies of amphipathic peptides. 相似文献
3.
Ethan S. Karp 《生物化学与生物物理学报:生物膜》2006,1758(6):772-780
Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca2+ regulator. 13C cross-polarization magic angle spinning spectra of 13C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding 13C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl 13C resonance in the corresponding NMR spectrum. 13C-15N REDOR solid-state NMR spectroscopic experiments revealed the distance between the 13C carbonyl carbon of Leu39 and the 15N amide nitrogen of Leu42 to be 4.2 ± 0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three 2H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11° ± 5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer. 相似文献
4.
Evgeniy Salnikov Philippe Bertani Jan Raap Burkhard Bechinger 《Journal of biomolecular NMR》2009,45(4):373-387
In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural
evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state
NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have
been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy
similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis
of 15N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols,
i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the 15N chemical shift tensor of this Cα-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues.
Here we present an experimental investigation on the 15N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for
di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled
using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted
amino acids. A 15N chemical shift-1H-15N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main
tensor elements for the structural analysis of this membrane peptide. 相似文献
5.
Mote KR Gopinath T Traaseth NJ Kitchen J Gor'kov PL Brey WW Veglia G 《Journal of biomolecular NMR》2011,51(3):339-346
Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the
lipid bilayer. The method consists of measuring 1H-15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer.
A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly
derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using
these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of
suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that
integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([1H,15N]-SE-PISEMA-PDSD). The incorporation of 2D 15N/15N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically
aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential
assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers. 相似文献
6.
Lactophoricin (LPcin), a component of proteose peptone (113–135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing 1H-15N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting 15N 1D and 2D 1H-15N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built 1H-15N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55–75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies. 相似文献
7.
Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes. 相似文献
8.
Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes. 相似文献
9.
10.
Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. 15N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. 15N longitudinal (R1) and transverse (R2) auto relaxation rates, heteronuclear 1H-15N steady-state NOEs, and transverse cross-relaxation rates (xy) were obtained at 30 °C for all resolved backbone secondary amide groups using 1 H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (m) is 5.09±0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D/D) is 0.81±0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1–19), the C-terminus (residues 68–73) and the loop connecting the first two -strands (residues 30–37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond–millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization. 相似文献
11.
Xi Cheng Sunhwan Jo Yifei Qi Francesca?M. Marassi Wonpil Im 《Biophysical journal》2015,108(8):1954-1962
Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys40, residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein dynamics but also protein-lipid interactions in detail. 相似文献
12.
Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na+ flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4)2(β2)3, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4)2(β2)3 TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. 2H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively 15N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the α4 and (α4)2(β2)3 samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (α4)2(β2)3 TM2. A small degree of wobbling motion of the helix axis occurred in the (α4)2(β2)3 TM2. In addition, a subset of the (α4)2(β2)3 TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10- 4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions. 相似文献
13.
Jarbas M. Resende Rodrigo M. Verly Christopher Aisenbrey Amary Cesar Philippe Bertani Dorila Piló-Veloso Burkhard Bechinger 《Biophysical journal》2014
Phylloseptin-1, -2, and -3 are three members of the family of linear cationic antimicrobial peptides found in tree frogs. The highly homologous peptides encompass 19 amino acids, and only differ in the amino acid composition and charge at the six most carboxy-terminal residues. Here, we investigated how such subtle changes are reflected in their membrane interactions and how these can be correlated to their biological activities. To this end, the three peptides were labeled with stable isotopes, reconstituted into oriented phospholipid bilayers, and their detailed topology determined by a combined approach using 2H and 15N solid-state NMR spectroscopy. Although phylloseptin-2 and -3 adopt perfect in-plane alignments, the tilt angle of phylloseptin-1 deviates by 8° probably to assure a more water exposed localization of the lysine-17 side chain. Furthermore, different azimuthal angles are observed, positioning the amphipathic helices of all three peptides with the charged residues well exposed to the water phase. Interestingly, our studies also reveal that two orientation-dependent 2H quadrupolar splittings from methyl-deuterated alanines and one 15N amide chemical shift are sufficient to unambiguously determine the topology of phylloseptin-1, where quadrupolar splittings close to the maximum impose the most stringent angular restraints. As a result of these studies, a strategy is proposed where the topology of a peptide structure can be determined accurately from the labeling with 15N and 2H isotopes of only a few amino acid residues. 相似文献
14.
Niels Johan Christensen Peter Ibsen Hansen Tore Folkerman Søren Balling Engelsen 《Carbohydrate research》2010,345(4):474-9509
The conformational and hydration properties of the two disaccharides methyl β-cellobioside and methyl β-laminarabioside were investigated by NMR spectroscopy and explicit solvation molecular dynamics simulations using the carbohydrate solution force field (CSFF). Adiabatic maps produced with this force field displayed 4 minima A: (Φ = 300°, Ψ = 280°), B: (Φ = 280°, Ψ = 210°), C: (Φ = 260°, Ψ = 60°), and D: (Φ = 60°, Ψ = 260°) for methyl β-cellobioside and 3 minima A: (Φ = 290°, Ψ = 130°), B: (Φ = 270°, Ψ = 290°), and C: (Φ = 60°, Ψ = 120°) for methyl β-laminarabioside. Molecular dynamics simulations were initiated from all minima. For each disaccharide, the simulation started from the A minimum was conducted for 50 ns, while the other minima were explored for 10 ns. The simulations revealed two stable minima for both compounds. For methyl β-cellobioside, the simulation minima in aqueous solution were shifted from their adiabatic map counterparts, while the simulation minima for methyl β-laminarabioside coincided with the corresponding adiabatic map minima. To validate the simulation results, NMR-derived NOEs and coupling constants across the glycoside linkage, 3JHC and 3JCH, were compared with values calculated from the MD trajectories. For each disaccharide, the best agreement was obtained for the simulations started at the A minimum. For both compounds, inter-ring water bridges in combination with the direct hydrogen bonds between the same groups were found to be determining factors for the overall solution structure of the disaccharides which differed from solid-state structures. Comparison with helical parameters showed that the preferred glycosidic dihedral configurations in the methyl β-cellobioside simulation were not highly compatible with the structure of cellulose, but that curdlan helix structures agreed relatively well with the methyl β-laminarabioside simulation. Polymers generated using glycosidic dihedral angles from the simulations revealed secondary structure motifs that that may help to elucidate polymer associations and small-molecule binding. 相似文献
15.
Aisenbrey C Bertani P Henklein P Bechinger B 《European biophysics journal : EBJ》2007,36(4-5):451-460
Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to
understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy
on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide
bonds and domains relative to the bilayer normal. Using this approach the 15N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the 2H solid-state NMR spectra acquired from peptides labelled with 3,3,3-2H3-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium
NMR line shapes are highly sensitive to small variations in the alignment of the Cα–Cβ bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to
the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field
direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state
of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic
helices. Thereafter investigations of the viral channel peptides Vpu1–27 and Influenza A M222–46 are shown. Whereas the 15N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate
considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation
times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and
oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously
been performed.
Dedicated to Prof. K. Arnold on the occasion of his 65th birthday. 相似文献
16.
Does Solid-state <Superscript>15</Superscript>N NMR Spectroscopy Detect all Soil Organic Nitrogen? 总被引:1,自引:1,他引:0
Virtually all of the N detected by 15N cross polarization (CP) NMR spectra of four HF-treated soil clay fractions is amide N. However, the intensity of this 15N CP NMR signal (per unit N) is 27–57% lower than detected for a wheat protein, gliadin. There are two possible explanations
– either the amide N in the soil clay fractions produces proportionately less NMR signal than does the amide N in gliadin,
or part of the N in the soil clay fractions produces little or no NMR signal. The cross polarization dynamics of the gliadin
amide resonance and amide resonances detected for the soil clay fractions are very similar and thus should produce similar
amounts of signal, ruling out the first possibility. Therefore up to half or even more of the organic N in these soil clay
fractions must be in a form that is insensitive to NMR detection. For a model compound (caffeine), non-protonated heterocyclic
N produced less than 20% of the signal of an equivalent amount of amide N in gliadin. Results from several 13C NMR techniques provide further evidence that much of the undetected N in the soil clay fractions may be heterocyclic. 相似文献
17.
Solid-state NMR (SSNMR) is a powerful technique to describe the orientations of membrane proteins and peptides in their native membrane bilayer environments. The deuterium (2H) quadrupolar splitting (DQS), one of the SSNMR observables, has been used to characterize the orientations of various single-pass transmembrane (TM) helices using a semistatic rigid-body model such as the geometric analysis of labeled alanine (GALA) method. However, dynamic information of these TM helices, which could be related to important biological function, can be missing or misinterpreted with the semistatic model. We have investigated the orientation of WALP23 in an implicit membrane of dimyristoylglycerophosphocholine by determining an ensemble of structures using multiple conformer models with a DQS restraint potential. When a single conformer is used, the resulting helix orientation (tilt angle (τ) of 5.6 ± 3.2° and rotation angle (ρ) of 141.8 ± 40.6°) is similar to that determined by the GALA method. However, as the number of conformers is increased, the tilt angles of WALP23 ensemble structures become larger (26.9 ± 6.7°), which agrees well with previous molecular dynamics simulation results. In addition, the ensemble structure distribution shows excellent agreement with the two-dimensional free energy surface as a function of WALP23's τ and ρ. These results demonstrate that SSNMR ensemble dynamics provides a means to extract orientational and dynamic information of TM helices from their SSNMR observables and to explain the discrepancy between molecular dynamics simulation and GALA-based interpretation of DQS data. 相似文献
18.
Rieko Ishima Kazuhiko Yamasaki Minoru Saito Kuniaki Nagayama 《Journal of biomolecular NMR》1995,6(2):217-220
Summary Spectral densities of the 15N amide in Escherichia coli ribonuclease HI, obtained from NMR relaxation experiments, were compared with those calculated using a molecular dynamics (MD) simulation. All calculations and comparisons assumed that the auto-correlation function describing the internal motions of the molecule was independent of the auto-correlation function associated with overall rotational diffusion. Comparisons were limited to those residues for which the auto-correlation function of internal motions rapidly relaxed and reached a steady state within 205 ps. The results show the importance of frequency components as well as amplitudes of internal motions in order to obtain a meaningful comparison of MD simulations with NMR data. 相似文献
19.
Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy
We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments
make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration.
We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange
rates negligible within the individual 15N–T
1 timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s−1. Backbone amide 15N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no
systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38,
N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical
exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D2O is employed as a solvent for sample preparation. Due to the intrinsically long 15N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate
regime that is not accessible so far with exchange quench and MEXICO type experiments. 相似文献
20.
In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide 15N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles. The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites. The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S
2 is on average 0.11 ± 0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of 1H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s10858-006-9008-y and is accessible for authorized users. 相似文献