Effects of cyclic GMP on the secondary structure of cyclic GMP dependent protein kinase and analysis of the enzyme's amino-terminal domain by far-ultraviolet circular dichroism

Far-UV circular dichroism spectra of bovine lung cyclic GMP dependent protein kinase (G-kinase) show that the enzyme contains alpha-helical and beta-pleated sheet elements. Binding of cyclic GMP changes the spectra in a way consistent with the induction of beta-sheet from random coil. Examination of the amino-terminal sequence of G-kinase indicates the presence of a strongly alpha-helical segment with several features in common with the leucine zipper motif. We propose that this sequence may be the important part of the dimerization domain of the enzyme. A synthetic peptide corresponding to amino acids 1-39 of G-kinase has a strongly alpha-helical CD spectrum, supporting the predicted secondary structure of this amino-terminal sequence. In contrast to the native enzyme, a structure reduced in alpha-helix was found when a constitutively active form of G-kinase, which lacks amino acids 1-77, was studied.     

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1,3)-Ac2-alpha-D-GalN3+ ++]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CalphaH chemical shift perturbations, 3JNH:CalphaH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.     

Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain HN protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the epsilon HN protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

Proton nuclear magnetic resonance study of an active pentapeptide fragment of ubiquitin

The aqueous solution conformation of Tyr-Asn-Ile-Gln-Lys (UB5) corresponding to positions 59-63 of the polypeptide, ubiquitin, has been investigated by proton NMR. Like the parent protein, UB5 induces nonspecifically both T and B lymphocyte differentiation. The various NH and CH resonances of this pentapeptide have been assigned, and its solution conformation has been probed through a study of chemical shift variations with pH, temperature dependence of amide hydrogen chemical shifts, vicinal NH--C alpha H and C alpha H--C beta H2 coupling constant data, and amide hydrogen-exchange rates. The latter were measured in H2O by using a combination of transfer of solvent saturation and saturation recovery NMR experiments. The data are compatible with the assumption of a highly motile dynamic equilibrium among different conformations for this peptide. The various secondary amide hydrogens remain essentially exposed to the solvent. The temperature-dependence study of the amide hydrogen chemical shifts also did not reveal any strong internal hydrogen bonds. A rotamer population analysis of tyrosine and asparagine side chains suggests that two of the rotomers are predominantly populated for each of these residues. From these results, a picture emerges of the dynamic conformation of UB5 in aqueous solution.     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain H^N protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the εH^N protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin.

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly alpha-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.     

Sequence-specific 1H NMR assignments and identification of slowly exchanging amide protons in murine epidermal growth factor

Proton nuclear magnetic resonance (1H NMR) assignments for the murine epidermal growth factor (mEGF) in aqueous solution were determined by using two-dimensional NMR at pH 3.1 and 28 degrees C. The assignments are complete for all backbone hydrogen atoms, with the exception of the N-terminal amino group, and for 46 of the 53 side chains. Among the additional seven amino acid residues, three have complete assignments for all but one side-chain proton, and between two and four protons are missing for the remaining four residues. The sequential assignments by nuclear Overhauser effect spectroscopy are consistent with the chemically determined amino acid sequence. The NMR data show that the conformations of both the Tyr3-Pro4 and Cys6-Pro7 peptide bonds are trans in the predominant solution structure. Proton-deuterium exchange rate constants were also measured for 13 slowly exchanging amide protons. The information presented here has been used elsewhere to determine the three-dimensional structure of mEGF in aqueous solution.     

All-atom molecular dynamics study of EAK16 peptide: the effect of pH on single-chain conformation,dimerization and self-assembly behavior

Single-chain equilibrium conformation and dimerization of the three types of ionic EAK16 peptide are studied under three pH conditions using all-atom molecular dynamics simulations. It is found that both the single-chain conformation and the dimerization process of EAK16-IV are considerably different from those of the two other types, EAK16-I and EAK16-II. The value of pH is found to have a stronger effect on the single-chain conformation and dimerization of EAK16-IV. It is shown that in addition to the charge pattern on the peptide chains, the size of the side chains of the charged amino acids plays role in the conformation of the peptide chains and their dimerization. The results shed light on the pH-dependent self-assembly behavior of EAK16 peptide in the bulk solution, which has been reported in the literature.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the beta-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant alpha-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular 31P-1H and 1H-1H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.     

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.     

The distal C-terminal region of the KcsA potassium channel is a pH-dependent tetramerization domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11)?M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH?5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested.     

Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy

The structure of the membrane anchor domain (VpuMA) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilayers by homonuclear two-dimensional and solid-state nuclear magnetic resonance spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu1-39 (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) solution, afford a compact well-defined U-shaped structure comprised of an initial turn (residues 1-6) followed by a linker (7-9) and a short helix on the N-terminal side (10-16) and a further longer helix on the C-terminal side (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded VpuMA is positioned. As the observed solution structure is inconsistent with the formation of ion-conductive membrane pores defined previously for VpuMA in planar lipid bilayers, the isolated VpuMA domain as peptide Vpu1-27 was investigated in oriented phospholipid bilayers by proton-decoupled 15N cross polarization solid-state NMR spectroscopy. The line widths and chemical shift data of three selectively 15N-labeled peptides are consistent with a transmembrane alignment of a helical polypeptide. Chemical shift tensor calculations imply that the data sets are compatible with a model in which the nascent helices of the folded solution structure reassemble to form a more regular linear alpha-helix that lies parallel to the bilayer normal with a tilt angle of 相似文献   

A transmembrane peptide from the human EGF receptor: behaviour of the cytoplasmic juxtamembrane domain in lipid bilayers

Solid state (2)H NMR spectroscopy was employed to study peptides related to the transmembrane domain of the human epidermal growth factor receptor, for insight into the interaction of its cytoplasmic juxtamembrane domain with the membrane surface. Since such receptors have clusters of (+)charged amino acids in this region, the effect of (-)charged phosphatidylserine at the concentration found naturally in the cytoplasmic leaflet (15 mol%) was considered. Each peptide contained 34 amino acids, which included the hydrophobic 23 amino acid stretch thought to span the membrane and a ten amino acid segment beyond the 'cytoplasmic' surface. Non-perturbing deuterium probe nuclei were located within alanine side chains in intramembranous and extramembranous portions. (2)H NMR spectra were recorded at 35 degrees C and 65 degrees C in fluid lipid bilayers consisting of (zwitterionic) 1-palmitoyl-2-oleoylphosphatidylcholine, with and without 15 mol% (anionic) phosphatidylserine. The cationic extramembranous portion of the receptor backbone was found to be highly rotationally mobile on a time scale of 10(-4)-10(-5) s in both types of membrane - as was the alpha-helical intramembranous portion. Deuterium nuclei in alanine side chains (-CD(3)) detected modest changes in peptide backbone orientation and/or dynamics related to the presence of 1-stearoyl-2-oleoylphosphatidylserine: in the case of the extramembranous portion of the peptide these seemed related to lipid charge. Temperature effects on the peptide backbone external to the membrane were qualitatively different from effects on the helical transmembrane domain - likely reflecting the different physical constraints on these peptide regions and the greater flexibility of the extramembranous domain. Effects related to lipid charge could be detected in the spectrum of CD(3) groups on the internally mobile side chain of Val(650), six residues beyond the membrane surface.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

An investigation into the native-like properties of de novo designed cavitand-based four-helix bundle proteins

We investigated the hydrophobic packing of two previously designed caviteins, LG2 and LG3, which differ by one Gly in the linker regions between the peptide sequence and the cavitand scaffold. We sought to diminish the putative native-like properties of LG2 and LG3, and see if we could diagnose a change in the conformational specificity of the hydrophobic core. We replaced the leucine residues with norleucine residues at the hydrophobic positions in LG2 and LG3, to create NG2 and NG3, respectively. LG2 exhibited more dispersion, but less sharp signals than LG3 in the amide region of its (1)H NMR spectrum. NG3 and NG2 were found to be slightly less helical and significantly less stable toward guanidine hydrochloride compared with their reference caviteins. The (1)H NMR spectrum of NG2 was very similar to that of LG2, whereas there was a noticeable loss in the number and sharpness of the amide signals of NG3 compared with LG3. These data suggest that LG3 is very well packed; a loss in conformational specificity resulted from replacement of the leucine residues with norleucine residues. In contrast, the packing and dynamics of the hydrophobic core in LG2 were similar to those in NG2 (both more modest than LG3), as their (1)H NMR spectra were virtually indistinguishable. Overall, substitution of leucine by norleucine provided an efficient, convenient, and informative probe of the packing and dynamics of our caviteins' hydrophobic cores.     

Structure and dynamics of melittin in lysomyristoyl phosphatidylcholine micelles determined by nuclear magnetic resonance.

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.