Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by 1H NMR spectroscopy

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. We have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using 1H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D2O at 24 degrees C were used to follow the exchange of the slowest amides in the protein. Multiple exponential fitting of the exchange data showed that these amides (29 +/- 3 at pH 4.5) exchanged in two kinetic sets with exchange rates [(1.2 +/- 0.4) x 10(-4) s-1 and (4.1 +/- 1.2) x 10(-7) s-1] that differed by more than 100-fold, the slower kinetic set being retarded 10(5)-fold relative to poly(DL-alanine). The exchange rate constant for the slowest set of amides exhibited an unusual pD dependence, being proportional to [OD-]1/2. It is shown that this is an artifact of the multiple exponential fitting of the data, and a new method of presentation of exchange data as a function of pD is introduced. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55 degrees C and that about 30 amides have exchange rates retarded by at least 10(5)-fold at 24 degrees C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. This is explained as being due to long-range electrostatic effects arising both from the protein itself and also from the anionic detergent molecules. Hydrogen exchange studies on the products of proteinase K digestion of the protein localized the slowly exchanging amides to the hydrophobic core of the protein. Relaxation [Henry, G.D., Weiner, J.H., & Sykes, B.D. (1986) Biochemistry 25, 590-598] and solid-state NMR experiments [Leo, G.C., Colnago, L.A., Valentine, K.G., & Opella, S.J. (1987) Biochemistry 26, 854-862] have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile.(ABSTRACT TRUNCATED AT 400 WORDS)     

Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Backbone dynamics of a model membrane protein: 13C NMR spectroscopy of alanine methyl groups in detergent-solubilized M13 coat protein

The filamentous coliphage M13 possesses multiple copies of a 50-residue coat protein which is inserted into the inner membrane of Escherichia coli during infection. 13C nuclear magnetic resonance (NMR) spectroscopy has been used to probe the structure and dynamics of M13 coat protein solubilized in detergent micelles. A comparison of backbone dynamics within the hydrophobic core region and the hydrophilic terminal domains was obtained by biosynthetic incorporation of [3-13C]alanine. Alanine is distributed throughout the protein and accounts for 10 residues (i.e., 20% of the total). Similar 13C NMR spectra of the protein have been obtained in two anionic detergents, sodium deoxycholate and sodium dodecyl sulfate, although the structures and physical properties of these solubilizing agents are quite different. The N-terminal alanine residues, assigned by pH titration, and the penultimate residue, assigned by carboxypeptidase A digestion, give rise to analogous peaks in both detergent systems. The pKa of Ala-1 (approximately 8.8) and the relaxation parameters of individual carbon atoms (T1, T2, and the nuclear Overhauser enhancement) are also generally similar, suggesting a similarity in the overall protein structure. Relaxation data have been analyzed according to the model-free approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall correlation times were obtained by fitting the three experimental relaxation values for a given well-resolved single carbon atom to obtain a unique value for the generalized order parameter, S2, and the effective correlation time, tau e. The former parameter reflects the spatial restriction of motion, and the latter, the rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Backbone dynamics of a model membrane protein: assignment of the carbonyl carbon 13C NMR resonances in detergent-solubilized M13 coat protein

The major coat protein of the filamentous bacteriophage M13 is a 50-residue amphiphilic polypeptide which is inserted, as an integral membrane-spanning protein, in the inner membrane of the Escherichia coli host during infection. 13C was incorporated biosynthetically into a total of 23 of the peptide carbonyls using labeled amino acids (alanine, glycine, lysine, phenylalanine, and proline). The structure and dynamics of carbonyl-labeled M13 coat protein were monitored by 13C nuclear magnetic resonance (NMR) spectroscopy. Assignment of many resonances was achieved by using protease digestion, pH titration, or labeling of the peptide bond with both 13C and 15N. The carbonyl region of the natural-abundance 13C NMR spectrum of M13 coat protein in sodium dodecyl sulfate solution shows approximately eight backbone carbonyl resonances with line widths much narrower than the rest. Three of these more mobile residues correspond to assigned peaks (glycine-3, lysine-48, and alanine-49) in the individual amino acid spectra, and another almost certainly arises from glutamic acid-2. A ninth residue, alanine-1, also gives rise to a very narrow carbonyl resonance if the pH is well above or below the pKa of the terminal amino group. These data suggest that only about four residues at either end of the protein experience large-amplitude spatial fluctuations; the rest of the molecule is essentially rigid on the time scale of the overall rotational tumbling of the protein-detergent complex. The relative exposure of different regions of detergent-bound protein was monitored by limited digestion with proteinase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignment of amide 1H and 15N NMR resonances in detergent-solubilized M13 coat protein: a model for the coat protein dimer.

The major coat protein of the filamentous coliphage M13 is a 50-residue integral membrane protein. Detergent-solubilized M13 coat protein is a promising candidate for structure determination by nuclear magnetic resonance methods as the protein can be prepared in large quantities and the protein-containing micelle is reasonably small. Under the conditions of our experiments, SDS-bound coat protein exists as a dimer with an apparent molecular weight of 27,000. Broad lines and poor resolution in the 1H spectrum have led us to adopt an 15N-directed approach, in which the coat protein was labeled both uniformly with 15N and selectively with [alpha-15N]alanine, -glycine, -valine, -leucine, -isoleucine, phenylalanine, -lysine, -tyrosine, and -methionine. Nitrogen resonances were assigned as far as possible using carboxypeptidase digestion, double-labeling, and an independent knowledge of the amide proton exchange rates determined from neighboring assigned 13C-labeled carbonyl carbons. 1H/15N heteronuclear multiple quantum coherence (HMQC) spectroscopy of both uniform and site-selectively-labeled proteins subsequently correlated amide nitrogen with amide proton chemical shifts, and the assignments were completed sequentially from homonuclear NOESY and HMQC-NOESY spectra. The most slowly exchanging amide protons were shown to occur in a continuous stretch extending from methionine-28 to phenylalanine-42. This sequence includes most of the resonances of the hydrophobic core, although it is shifted toward the C-terminal end of the protein. Strong NH to NH (i,i+1) nuclear Overhauser enhancements are a feature of the coat protein, which appears to be largely helical. Between 20 and 25 residues give rise to 2 juxtaposed resonances which can be seen clearly in the HMQC spectrum of uniform 15N-labeled coat protein. These residues are concentrated in a region extending from the beginning of the membrane-spanning sequence through to the disordered region near the C-terminus. We propose that dodecyl sulfate-bound M13 coat protein consists of two independent domains, an N-terminal helix which is in a state of moderately fast dynamic flux and a long, stable, C-terminal membrane-spanning helix, which undergoes extensive interactions with a second monomer. Amide 1H chemical shifts are consistent with this picture; in addition, a marked periodicity is observed at the C-terminal end of the molecule.     

Structure and dynamics of detergent-solubilized M13 coat protein (an integral membrane protein) determined by 13C and 15N nuclear magnetic resonance spectroscopy

The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection. M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies. An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments. The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei. Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics. A model is proposed for the structure of a coat protein dimer in detergent micelles.     

Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: use of the INEPT experiment to follow individual amides in detergent-solubilized M13 coat protein

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1H nuclear magnetic resonance (NMR) study [O'Neil, J. D. J., & Sykes, B. D. (1988) Biochemistry 27, 2753-2762], multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow "kinetic sets" containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10(5)-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein we use 15N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiment [Morris, G. A., & Freeman, R. (1979) J. Am. Chem. Soc. 101, 760-762] can be used to transfer magnetization to the 15N nucleus from a coupled proton; when 15N-labeled protonated protein is dissolved in 2H2O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H+ and OH- ions. Base catalysis is significantly more effective, resulting in a characteristic minimum rate in model peptides at pH approximately equal to 3. Rate versus pH profiles have been obtained by using the INEPT experiment for the amides of leucine-14, leucine-41, tyrosine-21, tyrosine-24, and valines-29, -30, -31, and -33 in M13 coat protein. The valine residues exchange most slowly and at very similar rates, showing an apparent 10(6)-fold retardation over poly(DL-alanine). A substantial basic shift in the pH of the minimum rate (up to 1.5 pH units) was also observed for some residues. Possible reasons for the shift include accumulation of catalytic H+ ions at the negatively charged micelle surface or destabilization of the negatively charged transition state of the base-catalyzed reaction by either charge or hydrophobic effects within the micelle. The time-dependent exchange-out experiment is suitable for slow exchange rates (kex), i.e., less than (1-2) x 10(-4) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Environmental modulation of M13 coat protein tryptophan fluorescence dynamics

The effects of detergent [deoxycholate (DOC) and phospholipid [dimyristoylphosphatidylcholine (DMPC)] environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay. The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method). This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC. The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments. The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion. The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1. In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component. Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit. The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and orientational information of the membrane embedded M13 coat protein by (13)C-MAS NMR spectroscopy

Oriented and unoriented M13 coat protein, incorporated into dimyristoyl phosphatidylcholine bilayers, has been studied by (13)C-magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Rotational resonance experiments provided two distance constraints between Calpha and C&z.dbnd6;O positions of the labelled residues Val-29/Val-30 (0.4+/-0.5nm) and Val-29/Val-31 (0.45+/-0. 5nm) in its hydrophobic domain. The derived dihedral angles (Phi, Psi) for Val-30 revealed a local alpha-helical conformation. (13)C-CP-MAS experiments on uniformly aligned samples (MAOSS experiments) using the (13)C&z.dbnd6;O labelled site of Val-30 allowed the determination of the helix tilt (20 degrees +/-10 degrees ) in the membrane. It is shown that one uniform MAS high-resolution solid state NMR approach can be used to obtain structural and orientational data.     

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a ¹⁵N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of ¹⁵N solid-state NMR spectroscopy (10⁻⁴ seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc.     

Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy

The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order in protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell.     

Conformation and aggregation of M13 coat protein studied by molecular dynamics.

Molecular dynamics (MD) simulations are performed on M13 coat protein, a small membrane protein for which both alpha- and beta-structures have been suggested. The simulations are started from initial conformations that are either monomers or dimers of alpha-helices or U-shaped beta-sheets. The lipid bilayer is represented by a hydrophobic potential. The results are analyzed in terms of stability, energy and secondary structure. The U-shaped beta-structure changes from a planar to a twisted form with larger twist for the monomer than the dimer. The beta-sheet is much more flexible than the alpha-helix as monitored by the root mean square (rms) fluctuations of the C alpha atoms. A comparison of the energies after 100 ps MD simulation shows that of the monomers, the alpha-helix has the lowest energy. The energy difference between alpha- and beta-structures decreases from 266 kJ/mol to 148 kJ/mol, when going from monomers to dimers. It is expected that this difference will decrease with higher aggregation numbers.     

Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and mixed bilayers

Coat protein of bacteriophage M13 is examined in micelles and vesicles by time-resolved tryptophan fluorescence and anisotropy decay measurements and circular dichroism experiments. Circular dichroism indicates that the coat protein has alpha-helix (60%) and beta-structure (28%) in 700 mM sodium dodecyl sulfate micelles and predominantly beta-structure (94%) in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidic acid (80/20 w/w) small unilamellar vesicles. The fluorescence decay at 344 nm of the single tryptophan in the coat protein after excitation at 295 or 300 nm is a triple exponential. In the micelles the anisotropy decay is a double exponential. A short, temperature-independent correlation time of 0.5 +/- 0.2 ns reflects a rapid depolarization process within the coat protein. The overall rotation of the coat protein-detergent complex is observed in the decay as a longer correlation time of 9.8 +/- 0.5 ns (at 20 degrees C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In vesicles at all lipid to protein molar ratios in the range from 20 to 410, the calculated order parameter is constant with a value of 0.7 +/- 0.1 from 10 to 40 degrees C, although the lipids undergo the gel to liquid-crystalline phase transition. The longer correlation time decreases gradually on increasing temperature. This effect probably arises from an increasing segmental mobility within the coat protein. The results are consistent with a model in which the coat protein has a beta-structure and the tryptophan indole rings do not experience the motion of the lipids in the bilayer because of protein-protein aggregation.     

Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.     

Detergent-solubilized M13 coat protein exists as an asymmetric dimer. Observation of individual monomers by 15N, 13C and 1H nuclear magnetic resonance spectroscopy

M13 coat protein is a simple integral membrane protein isolated from the filamentous coliphage M13. Isotopic labels (13C and 15N) may be incorporated biosynthetically into the protein backbone. 13C nuclear magnetic resonance spectroscopy of carbonyl carbon atoms and two-dimensional 1H-detected 15N-1H heteronuclear shift correlation of coat protein in dodecylsulphate micelles have shown many residues throughout the protein to give rise to two distinct resonances of equal intensity. Chemical shift differences between the two forms are small, indicating the existence of two slightly different but equally populated conformational states. We suggest that the two conformers correspond to the inequivalent monomers of an asymmetric coat protein dimer and propose a mechanism for the generation of such a dimer.     

Side-chain conformations in an unfolded protein: chi1 distributions in denatured hen lysozyme determined by heteronuclear 13C, 15N NMR spectroscopy.

Using a 13C and 15N-labelled sample, multi-dimensional heteronuclear NMR techniques have been carried out to characterise hen lysozyme denatured in 8 M urea at pH 2.0. The measurement of 3J(C',Cgamma) and 3J(N,Cgamma) coupling constants has enabled side-chain chi1 torsion angle populations to be probed in the denatured polypeptide chain. Analysis of the coupling constant data has allowed the relative populations of the three staggered rotamers about chi1 to be defined for 51 residues. The amino acids can broadly be divided into five classes that show differing side-chain conformational preferences in the denatured state. These range from a strong preference for the -60 degrees chi1 rotamer for methionine and leucine (74-79 % population) to a favouring of the +60 degrees chi1 rotamer for threonine (67 % population). The differences in behaviour reflect the steric and electrostatic characteristics of the side-chains concerned. A close agreement is seen between the chi1 populations calculated from the experimental coupling constant data and predictions from the statistical model for a random coil that uses the chi1 torsion angle distributions in a data base of native protein structures. Short-range interactions therefore dominate in determining the local conformational properties of side-chains in a denatured protein. Deviations are, however, observed for many of the aromatic residues involved in hydrophobic clusters within the denatured protein. For these residues the effects of additional non-local interactions in the clusters presumably play a major role in determining the chi1 preferences.     

Comparison of the structure and dynamics of chicken gizzard and rabbit cardiac tropomyosins: 1H NMR spectroscopy and measurement of amide hydrogen exchange rates

The side chain and backbone mobilities of chicken gizzard tropomyosin (TM) and its nonpolymerizable derivative have been investigated by H NMR spectroscopy and amide hydrogen exchange kinetics and compared to those of rabbit cardiac TM and its nonpolymerizable derivative. Analysis of the 300-MHz H NMR spectra of native chicken gizzard and rabbit cardiac TMs and their nonpolymerizable derivatives showed that the line widths of the aromatic and histidine residues were within a factor of 2 for all four proteins, demonstrating that the side chain mobility of these residues is similar in the different TMs. Direct proton exchange-out kinetics were determined in D2O in the pD range 1.5-3.0 at 25 degrees C by H NMR spectroscopy. Multiple exponential fitting of the exchange data indicated the presence in gizzard TM of at least three kinetically distinct classes of amide hydrogens at pD 1.7 with average population sizes of 147, 74, and 61, whose rates were retarded by a factor of 10, 10(3), and 10(5), respectively, relative to the random-coil peptide poly(DL-alanine). Measurement of the direct exchange kinetics of both rabbit cardiac and nonpolymerizable gizzard TMs showed that their rate constants and population sizes were within experimental error of those for the gizzard protein, except that the fast exchanging class for cardiac TM was increased in size while that of the nonpolymerizable gizzard TM was reduced, relative to that for gizzard TM. Comparison of the exchange-out kinetics for the cardiac and gizzard proteins at pH 2.0 and 55 degrees C, where only the two slowly exchanging amide hydrogen sets are measured, again demonstrated the similarity of their kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy

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 C_α–C_β 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 Vpu_1–27 and Influenza A M2_22–46 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.     

1H NMR dipolar echo decay spectroscopy: a sensitive probe of membrane structure

The structural and motional properties of membrane lipids in various physical states and macroscopic organizations are elucidated by 1H NMR dipolar echo decay spectroscopy (DECODE). Multilamellar lipid dispersions in the gel (L beta') and liquid-crystalline (L alpha) states and a nonbilayer, hexagonal (HII) phase are readily distinguished, a dynamic profile within these phases is identified, and dipolar order parameters are obtained in the fluid phases. The method is suitable for any pulsed NMR spectrometer. DECODE provides the first depth-dependent assay of lipid order that does not rely on isotopic labeling or exogenous probe.