Assessing the effects of time and spatial averaging in 15N chemical shift/15N-1H dipolar correlation solid state NMR experiments

The effect of time and spatial averaging on ¹⁵N chemical shift/¹H-¹⁵N dipolar correlation spectra, i.e., PISEMA spectra, of -helical membrane peptides and proteins is investigated. Three types of motion are considered: (a) Librational motion of the peptide planes in the -helix; (b) rotation of the helix about its long axis; and (c) wobble of the helix about a nominal tilt angle. A 2ns molecular dynamics simulation of helix D of bacteriorhodopsin is used to determine the effect of librational motion on the spectral parameters. For the time averaging, the rotation and wobble of this same helix are modelled by assuming either Gaussian motion about the respective angles or a uniform distribution of a given width. For the spatial averaging, regions of possible ¹⁵N chemical shift/¹H-¹⁵N dipolar splittings are computed for a distribution of rotations and/or tilt angles of the helix. The computed spectra show that under certain motional modes the ¹⁵N chemical shift/¹H-¹⁵N dipolar pairs for each of the residues do not form patterns which mimic helical wheel patterns. As a result, the unambiguous identification of helix tilt and helix rotation without any resonance assignments or on the basis of a single assignment may be difficult.     

Lysine side-chain dynamics derived from 13C-multiplet NMR relaxation studies on di- and tripeptides

Summary ¹³C NMR relaxation data have been used to determine dipolar auto- and cross-correlation times for the di- and tripeptides GK, KG and GKG, primarily to analyze lysine side-chain motional dynamics. In general, correlation times are largest for backbone positions and decrease on going through the lysine side chain, consistent with the idea of increased mobility at C and C methylenes. Correlation times, however, vary with the peptide ionization state. In the zwitterionic state of GK, for example, both auto-and cross-correlation times are at their maximum values, indicating reduced internal motions probably resulting from intramolecular electrostatic interactions. Modifying the charge state increases motional fluctuations. Activation energies determined from the temperature dependence of CH rotational autocorrelation times at neutral pH are approximately equal for glycine and lysine C and lysine C and C positions (4.1±0.2 to 4.5±0.2 kcal/mol) and tend to decrease slightly for lysine C and C (3.8±0.2 to 4.3±0.2 kcal/mol). The sign of lysine side-chain cross-correlations could not be explained by using any available rotational model, including one parameterized for multiple internally restricted rotations and anisotropic overall tumbling. Molecular and stochastic dynamics calculations were performed to obtain insight into correlated internal rotations and coupled overall tumbling and internal motions. Relatively strong correlations were found for i,i+1 backbone and lysine side-chain internal bond rotations. Stochastic dynamics calculations were more successful at explaining experimentally observed correlation times. In the fully charged state, a preferred conformation was detected with an all-trans lysine side chain.Abbreviations rf radio frequency - GK dipeptide glycine-lysine - KG dipeptide lysine-glycine - GKG tripeptide glycine-lysine-glycine     

Spin label and 2H-NMR studies on the interaction of melanotropic peptides with lipid bilayers

The interaction of the cationic tridecapeptide -melanocyte stimulating hormone (-MSH) and the biologically more active analog [Nle⁴, DPhe⁷]--MSH with lipid membranes was investigated by means of ESR of spin probes incorporated in the bilayer, and NMR of deuterated lipids. All spin labels used here, stearic acid and phospholipid derivatives labeled at the 5^th and 12^th position of the hydrocarbon chain, and the cholestane label, incorporated into anionic vesicles of DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) in the liquid-crystalline phase, indicated that both peptides decrease the motional freedom of the acyl chains. No peptide effect was detected with neutral lipid bilayers. Changes in the -deuteron quadrupolar splittings and spin lattice relaxation time of DMPG deuterated at the glycerol headgroup paralleled the results obtained with ESR, showing that the peptides cause a better packing both at the headgroup and at the acyl chain bilayer regions. The stronger effect caused by the more potent analog in the membrane structure, when compared to the native hormone, is discussed in terms of its larger lipid association constant and/or its deeper penetration into the bilayer.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

Backbone dynamics of bacteriorhodopsin as studied by <Superscript>13</Superscript>C solid-state NMR spectroscopy

The surface dynamics of bacteriorhodopsin was examined by measurements of site-specific ¹³C–¹H dipolar couplings in [3-¹³C]Ala-labeled bacteriorhodopsin. Motions of slow or intermediate frequency (correlation time <50 µs) scale down ¹³C–¹H dipolar couplings according to the motional amplitude. The two-dimensional dipolar and chemical shift (DIPSHIFT) correlation technique was utilized to obtain the dipolar coupling strength for each resolved peak in the ¹³C MAS solid-state NMR spectrum, providing the molecular order parameter of the respective site. In addition to the rotation of the Ala methyl group, which scales the dipolar coupling to 1/3 of the rigid limit value, fluctuations of the C–C vector result in additional motional averaging. Typical order parameters measured for mobile sites in bacteriorhodopsin are between 0.25 and 0.29. These can be assigned to Ala103 of the C–D loop and Ala235 at the C-terminal -helix protruded from the membrane surface, and Ala196 of the F–G loop, as well as to Ala228 and Ala233 of the C-terminal -helix and Ala51 from the transmembrane -helix. Such order parameters departing significantly from the value of 0.33 for rotating methyl groups are obviously direct evidence for the presence of fluctuation motions of the Ala C–C vectors of intact preparations of fully hydrated, wild-type bacteriorhodopsin at ambient temperature. The order parameter for Ala160 from the expectantly more flexible E–F loop, however, is unavailable under highest-field NMR conditions, probably because increased chemical shift anisotropy together with intrinsic fluctuation motions result in an unresolved ¹³C NMR signal.     

Conformational isomerism and hydrogen-bonded motifs of anion assisted supramolecular self-assemblies using Cu/Co salts and pyridine-4-acetamide

Six novel metal-organic complex assemblies constructed from a conformation-flexible ligand - pyridine-4-acetamide (PAT) and inorganic Cu^II and Co^II salts have been synthesized and structurally characterized by single crystal X-ray diffraction analysis. Crystal structure analysis reveals five types of architectures by variation of metal salts. In {[Cu(PAT)₂Cl₂]}_n (1) and {[Co(PAT)₂Cl₂]}_n (3), PAT ligands bridge metal centers to form one-dimensional chains. The chains are extended to three dimensions with the aid of two types of hydrogen bonded motifs () and (12)). {[Cu(PAT)₂(NO₃)](NO₃)(THF)}_n (5) which exhibits two-dimensional coordinating layers forms open channels filled with solvent molecules. In [Cu(PAT)₂Cl₂] (2), [Co(PAT)₂Cl₂] (4) and [Co(PAT)₄(H₂O)₂](NO₃)₂(THF)₂ (6), PAT is observed as a monofunctional ligand. Complex 2 forms one-dimensional hydrogen bonded chains. Crystal structure of complex 4 has a two-dimensional infinite hydrogen-bonded network with and motifs formed by complementary amide-amide hydrogen bonds. [Co(PAT)₄(H₂O)₂](NO₃)₂(THF)₂ (6) crystallizes in centrosymmetric I4₁/a space group. Complex 6 forms chiral channels which are filled with twisted solvent helices and anion helices. Within each channel the solvent helix and the anion helix have the same handedness; and adjacent channels have opposite handedness. Complexes 1, 2 and complexes 3, 4 illustrate examples of conformational supramolecular isomerism in {[Cu(PAT)₂Cl₂]}_∞ and {[Co(PAT)₂Cl₂]}_∞, respectively. In these complexes, changes of PAT conformations and coordination geometry of metal center induced the structural versatility.     

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ¹³C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ¹³C=O isotope-labeled (Ala-Gly-Gly)₁₀. The torsion angles were determined to be (, ) = (–90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 3₁ helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3₁ helix of (Ala-Gly-Gly)₁₀ does not change by formic acid treatment although (Ala-Gly)₁₅ easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3₁ helix conformation of (Ala-Gly-Gly)₁₀ is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)₁₀ were also determined as (, ) = (–90°, 150° ) and this peptide is also considered to take 3₁ helix conformation.     

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW^5,19ALP23 (acetyl-GGALW⁵(LA)₆LW¹⁹LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific ²H- and ¹⁵N-labeled residues. GW^5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as ²H quadrupolar or ¹⁵N-¹H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW^5,19ALP23, the modified GW^4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the ²H and ¹⁵N NMR signals confirm that the extent of dynamic averaging, particularly rotational “slippage” about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional ²H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.     

Structure refinement of flexible proteins using dipolar couplings: Application to the protein p8MTCP1

The present study deals with the relevance of using mobility-averaged dipolar couplings for the structure refinement of flexible proteins. The 68-residue protein p8^MTCP1 has been chosen as model for this study. Its solution state consists mainly of three -helices. The two N-terminal helices are strapped in a well-determined -hairpin, whereas, due to an intrinsic mobility, the position of the third helix is less well defined in the NMR structure. To further characterize the degrees of freedom of this helix, we have measured the dipolar coupling constants in the backbone of p8^MTCP1 in a bicellar medium. We show here that including D _HN ^dip dipolar couplings in the structure calculation protocol improves the structure of the -hairpin but not the positioning of the third helix. This is due to the motional averaging of the dipolar couplings measured in the last helix. Performing two calculations with different force constants for the dipolar restraints highlights the inconstancy of these mobility-averaged dipolar couplings. Alternatively, prior to any structure calculations, comparing the values of the dipolar couplings measured in helix III to values back-calculated from an ideal helix demonstrates that they are atypical for a helix. This can be partly attributed to mobility effects since the inclusion of the ¹⁵N relaxation derived order parameter allows for a better fit.     

Calcium gating of H+ fluxes in chloroplasts affects acid-base-driven ATP formation

In previous work, calcium ions, bound at the lumenal side of the CF₀H⁺ channel, were suggested to keep a H⁺ flux gating site closed, favoring sequestered domain H⁺ ions flowing directly into the CF₀-CF₁ and driving ATP formation by a localized gradient. Treatments expected to displace Ca⁺⁺ from binding sites had the effect of allowing H⁺ ions in the sequestered domains to equilibrate with the lumen, and energy coupling showed delocalized characteristics. The existence of such a gating function implies that a closed-gate configuration would block lumenal H⁺ ions from entering the CF₀-CF₁ complex. In this work that prediction was tested using as an assay the dark, acid-base jump ATP formation phenomenon driven by H⁺ ions derived from succinic acid loaded into the lumen.Chlorpromazine, a photoaffinity probe for many proteins having high-affinity Ca⁺⁺-binding sites, covalently binds to the 8-kDa CF₀ subunit in the largest amounts when there is sufficient Ca⁺⁺ to favor the localized energy coupling mode, i.e., the gate closed configuration. Photoaffinity-bound chlorpromazine blocked 50% or more of the succinate-dependent acid-base jump ATP formation, provided that the ionic conditions during the UV photoaffinity treatment were those which favor a localized energy coupling pattern and a higher level of chlorpromazine labeling of the 8-kDa CF₀ subunit. Thylakoids held under conditions favoring a delocalized energy coupling mode and less chlorpromazine labeling of the CF₀ subunit did not show any inhibition of acid-base jump ATP formation.Chlorpromazine and calmidazolium, another Ca⁺⁺-binding site probe, were also shown to block redox-derived H⁺ initially released into sequestered domains from entering the lumen, at low levels of domain H⁺ accumulation, but not at higher H⁺ uptake levels; ie., the closed gate state can be overcome by sufficiently acidic conditions. That is consistent with the observation that the inhibition of lumenal succinate-dependent ATP formation by photoaffinity-attached chlorpromazine can be reversed by lowering the pH of the acid stage from 5.5 to 4.5.The evidence is consistent with the concept that Ca⁺⁺ bound at the lumenal side of the CF₀ H⁺ channel can block H⁺ flux from either direction, consistent with the existence of a molecular structure in the CF₀ complex having the properties of a gate for H⁺ flux across the inner boundary of the CF₀. Such a gate could control the expression of localized or delocalized energy coupling gradients.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Domain orientation in β-cyclodextrin-loaded maltose binding protein: Diffusion anisotropy measurements confirm the results of a dipolar coupling study

Maltose binding protein (MBP) is a 370-residue two-domain molecule involved in bacterial chemotaxis and sugar uptake. Rotational diffusion tensors were calculated for a complex between MBP and -cyclodextrin using backbone ¹⁵N T₁ and T₁ relaxation times and steady state ¹H-¹⁵N NOE values. The tensors obtained for each of the two domains in the protein were subsequently used to determine the relative domain orientation in the molecule. The average domain orientation determined using this approach agrees well with results from dipolar coupling data, but differs significantly from the domain orientation deduced from X-ray studies of the complex.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.     

The dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by18O labeling of guanine nucleotide α-phosphoryls

The rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measuted in intact neuroblastoma N1E-115 cells by determining rates of¹⁸O incorporation from¹⁸O-water into the -phosphoryls of guanine and adenine nucleotides. The basal rate of guanine nucleotide -phosphoryl labeling ranged from 180 to 244 pmol·mg protein^–1·min^–1. Sodium nitroprusside (SNP) caused a sustained 3,4-fold increase in this¹⁸O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This¹⁸O-labeling rate (795 pmol·mg protein^–1·min^–1) corresponded with the sum of the low (1.7 M) and high (34 M) K_m phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol·mg protein^–1·min^–1 to which the highK _m species contributed 84%. This information and the characteristics of the profile of¹⁸O-labeled molecular species indicate that cGMP metabolism was restricted to a very discrete cellular compartment(s) of approximately 12% of the cell volume. Carbachol (1 mM) produced a transient increase (6-fold) in cellular cGMP concentration and a transient increase (90%) in the rate of¹⁸O labeling of -GTP during the first minute of treatment which translates into 30 additional cellular pools of cGMP hydrolyzed in this period. IBMX (1 mM) produced a relatively rapid increase in cellular cGMP (3- to 5-fold) and cAMP (2-fold) concentrations and a delayed inhibition of¹⁸O labeling of guanine and adenine nucleotide -phosphoryls without further elevation of cyclic nucleotide levels. These results indicate that besides inhibiting cyclic nucleotide hydrolysis, IBMX also imparts a time-dependent inhibitory influence on the generation of cyclic nucleotides. The data obtained show that measurement of¹⁸O labeling of guanine and adenine nucleotide -phosphoryls combined with measurements of cyclic nucleotide steady state levels provides a means to assess the rates of cyclic nucleotide synthesis and hydrolysis within intact cells and to identify the site(s) of action of agents that alter cellular cyclic nucleotide metabolism.Special Issue dedicated to Dr. O. H. Lowry.     

13C-NMR Investigation of the insertion of the bee venom melittin into lecithin vesicles

The orientation of melittin in lecithin membranes was investigated by means of ¹³C-NMR spectroscopy. Phospholipase-free melittin was labeled with ¹³C-methyl groups at the -amino side chains of lysine 7, 21, and 23. From the pH dependence of the corresponding ¹³C resonances, pK values of the lysine residues were derived that were different for free and membrane-bound melittin. The shift reagent Pr(NO₃)₃ induced shifts in the ¹³C resonance position of all three lysines when melittin and the shift reagent were added to a lecithin vesicle suspension, whereas Pr³⁺ ions included in the inner volume of the vesicles did not affect the ¹³C resonances of melittin bound to the outer vesicle membrane. A wedge-like structure was derived for the membrane-bound melittin, the lysine side chains of which are freely accessible to the aqueous solvent.Abbreviation NOE Nuclear Overhauser Enhancement     

NMR solution structure and dynamics of motilin in isotropic phospholipid bicellar solution

The structure and dynamics of the gastrointestinal peptide hormone motilin, consisting of 22 amino acid residues, have been studied in the presence of isotropic q=0.5 phospholipid bicelles. The NMR solution structure of the peptide in acidic bicelle solution was determined from 203 NOE-derived distance constraints and six backbone torsion angle constraints. Dynamic properties for the ¹³C-¹H vector in Leu10 were determined for motilin specifically labeled with ¹³C at this position by analysis of multiple-field relaxation data. The structure reveals an ordered -helical conformation between Glu9 and Lys20. The N-terminus is also well structured with a turn resembling that of a classical -turn. The ¹³C dynamics clearly show that motilin tumbles slowly in solution, with a correlation time characteristic of a large object. It was also found that motilin has a large degree of local flexibility as compared with what has previously been reported in SDS micelles. The results show that motilin interacts with the bicelle, displaying motional properties of a peptide bound to a membrane. In comparison, motilin in neutral bicelles seems less structured and more flexible. This study shows that the small isotropic bicelles are well suited for use as membrane-mimetic for structural as well as dynamical investigations of membrane-bound peptides by high-resolution NMR.     

Primary structure of the major glycan from human seminal transferrin

Human seminal transferrin (HSmT) is an iron-containing glycoprotein whose structural properties have not been adequately investigated. The carbohydrate content of the purified glycoprotein amount to 6.1%, and monosaccharide analysis revealed the major oligosaccharide moiety to be of the N-glycoside type. The carbohydrate chains were released from the iron-free form by digestion with peptide N-glycosidase F (PNGase F) in the presence of detergents such as SDS and-octylglucoside. After ethanol precipitation and fractionation on Bio-Gel P-6 and Bio-Gel P-2, the oligosaccharide was further purified on Mono-Q and desalted on Bio-Gel P-2. By 600-MHz¹H-NMR spectroscopy, the primary structure of the major N-linked oligosaccharide component was established to be: Abbreviations used HSmT human seminal transferrin - HSrT human serum transferrin - PNGase F peptide-N⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52), commonly known as peptide N-glycosidase F - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - GLC gas-liquid chromatography - FPLC fast liquid protein chromatography - EDTA ethylenediaminetetraacetic acid, disodium salt - PMFS phenylmethylsulfonyl fluoride - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man, Gal galactose - Fuc fucose     

The intramolecular δ<Superscript>15</Superscript>N of lysine responds to respiratory status in <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis>

Summary. Presented here is the first experimental evidence that natural, intramolecular, isotope ratios are sensitive to physiological status, based on observations of intramolecular δ¹⁵N of lysine in the mitochondrial mimic Paracoccus denitrificans. Paracoccus denitrificans, a versatile, gram-negative bacterium, was grown either aerobically or anaerobically on isotopically-characterized ammonium as sole cell-nitrogen source. Nitrogen isotope composition of the biomass with respect to source ammonium was = −6.2 ± 1.2‰ for whole cells under aerobic respiration, whereas cells grown anaerobically produced no net fractionation ( = −0.3 ± 0.23‰). Fractionation of ¹⁵N between protein nitrogen and total cell nitrogen increased during anaerobic respiration and suggests that residual nitrogen-containing compounds in bacterial cell membranes are isotopically lighter under anaerobic respiration. In aerobic cells, the lysine intramolecular difference between peptide and sidechain nitrogen is negligible, but in anaerobic cells was a remarkable Δ¹⁵N_{p − s} = δ¹⁵N_peptide − δ¹⁵N_sidechain = +11.0‰, driven predominantly by enrichment at the peptide N. Consideration of known lysine pathways suggests this to be likely due to enhanced synthesis of peptidoglycans in the anaerobic state. These data indicate that distinct pathway branching ratios associated with microbial respiration can be detected by natural intramolecular Δδ¹⁵N measurements, and are the first in vivo observations of position-specific measurements of nitrogen isotope fractionation.     

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na⁺/ HCO ₃ ^? cotransporter. We investigated whether the activity of Na⁺/HCO ₃ ^? cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased ²²Na uptake in the presence of HCO₃ but not in the presence of gluconate, indicating that trypsin does not alter diffusive ²²Na uptake but directly stimulates the Na⁺/HCO ₃ ^? cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na⁺/HCO ₃ ^? cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na⁺/HCO ₃ ^? cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na⁺/HCO ₃ ^? cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na⁺/HCO ₃ ^? cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S?). With the addition of fraction S?, the CSU of cAMP-PKA failed to inhibit the Na⁺/HCO ₃ ^? cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na⁺/HCO ₃ ^? cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na⁺/HCO ₃ ^? cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.     

Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear 1H−15N NMR spectroscopy

Summary The ¹⁵N relaxation rates of the -aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional ¹H–¹⁵N NMR spectroscopy. Measurements of ¹⁵N longitudinal (R_N(N_z)) and transverse (R_N(N_x,y)) relaxation rates and the {¹H} ¹⁵N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the ¹H–¹⁵N lingitudinal two-spin order, R_NH(2H _infZ ^supN N_Z), two-spin antiphase coherence, R_NH(2H _infN ^supZ N_x,y), and the proton longitudinal relaxation rate, R_H(H _infN ^supZ ), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide ¹⁵N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the core regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the frayed C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.To whom correspondence should be addressed.