Conformational studies of the N-terminal lipid-associating domain of human apolipoprotein C-I by CD and 1H NMR spectroscopy.

A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was synthesized using solid-phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC-I(1-38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS-bound apoC-I(1-38) was calculated from 464 NOE-based distance restraints using distance geometry methods. ApoC-I(1-38) adopts a helical structure between residues V4 and K30 and an extended C-terminus from Q31 when associated with SDS. The region K12-G15 undergoes slow conformational exchange as indicated by above-average amide resonance linewidths, large temperature coefficients, and fast exchange (< 2 h) of backbone amide protons with deuterium. The mobility of K12-G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC-I(1-38) is curved toward its hydrophobic face with bends of 125 degrees, centered at K12/E13, and 150 degrees, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC-I(1-38) and the previously obtained structure of the fragment apoC-I(35-53), we propose the secondary structure of intact apolipoprotein C-I.     

Conformation of microtubule-bound paclitaxel determined by fluorescence spectroscopy and REDOR NMR

The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 +/- 2 A. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the Stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a "hydrophobic collapsed" conformation in protic solvents and an "extended" conformation in aprotic solvents. The Stokes shift of microtubule-bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with (19)F at the para position of the C-2 benzoyl substituent and with (13)C and (15)N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 +/- 0.5 A, and the distance between the fluorine atom and the 3'-methine carbon was 10. 3 +/- 0.5 A. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.     

Heteronuclear NMR studies of human serum apolipoprotein A-I. Part I. Secondary structure in lipid-mimetic solution

Class I aminoacyl-tRNA synthetases have been thought to be single polypeptide enzymes. However, the complete genome sequence of a hyper thermophile Aquifex aeolicus suggests that the gene for leucyl-tRNA synthetases (LeuRS) is probably split into two pieces (leuS and leuS'). In this research, each gene was separately cloned and overexpressed in Escherichia coli and the protein products were examined for LeuRS activity. Leucylation activity was detected only when both gene products coexisted. Gel filtration analysis showed that the active form of A. aeolicus LeuRS has a heterodimeric (alpha/beta type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases.     

Conformation of sarafotoxin-6b in aqueous solution determined by NMR spectroscopy and distance geometry

The solution structure of sarafotoxin-6b in water has been determined using high-resolution NMR spectroscopy. 127 proton-proton distance measurements and three phi dihedral angle constraints derived from NMR spectra were used to calculate the solution structure using a combination of distance geometry and restrained molecular dynamics. The major structural feature of the resulting family of five structures was a right-handed alpha-helix extending from K9 to Q17. In contrast, the C-terminal region of the peptide appears not to adopt a preferred conformation in aqueous solution. The present structure is compared with those previously determined for endothelin peptides in non-aqueous solvents.     

The interaction of human apolipoprotein C-I with sub-micellar phospholipid.

Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture. The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid. Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM). Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules.     

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy.

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.     

Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR.

The dinucleoside phosphodiester dTdA is a slow substrate of staphylococcal nuclease (kcat = 3.8 X 10(-3) s-1) that forms binary E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. The enzyme enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of six proton resonances of dTdA, and these effects are abolished by binding of the competitive inhibitor 3',5'-pdTp. From paramagnetic effects of Co2+ on 1/T2 of phosphorus, koff of dTdA from the ternary E-Co(2+)-dTdA complex is greater than or equal to 4.8 X 10(4) s-1 and kon greater than or equal to 1.4 X 10(6) M-1 s-1, indicating the 1/T1 values to be in fast exchange. From paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with use of a correlation time of 1.6 ps on the basis of 1/T1 values at 250 and 600 MHz, 7 metal-nucleus distances and 9 lower-limit metal-nucleus distances are calculated. The long Co2+ to 31P distance of 4.1 +/- 0.9 A, which is intermediate between that expected for direct phosphoryl coordination (3.31 +/- 0.02 A) and a second sphere complex with an intervening water ligand (4.75 +/- 0.02 A), suggests either a distorted inner sphere complex or the rapid averaging of 18% inner sphere and 82% second sphere complexes and may explain the reduced catalytic activity with small dinucleotide substrates. Seventeen interproton distances and 108 lower limit interproton distances in dTdA in the ternary E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. While metal-substrate and interproton distances alone did not yield a unique structure, the combination of both sets of distances yielded a very narrow range of conformations for enzyme-bound dTdA, which was highly extended, with no base stacking, with high-anti glycosidic torsional angles for dT (64 degrees less than or equal to chi less than or equal to 73 degrees) and dA (66 degrees less than or equal to chi less than or equal to 68 degrees) and predominantly C-2'-endo sugar puckers for both nucleosides. Although the individual nucleosides are like those of B-DNA, their unstacked conformation, which is inappropriate for base pairing, as well as the conformational angles alpha and gamma of dA and zeta of dT, rule out B-DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation of thymosin β9 in water/fluoroalcohol solution determined by NMR spectroscopy

The conformation of thymosin β₉ in solution of 40% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol-d₂ in water has been investigated by two-dimensional ¹H-nmr spectroscopy. Under this condition thymosin β₉ adopts an ordered structure. The determination of the conformation of the peptide was based on a set of 304 approximate interproton distance constraints derived from nuclear Overhauser enhancement measurements. The conformation of thymosin β₉ includes two helical regions from residues 4 to 27 and 32 to 41. The two helices are separated by a poorly defined loop region between amino acids 28 and 31; the N-terminus of thymosin B₉ shows random-coil structure only. © 1997 John Wiley & Sons, Inc. Biopoly 41: 623–634, 1997     

Expression and purification of recombinant human apolipoprotein C-I in Pichia pastoris

Apolipoprotein C-I (ApoC-I) is a small, basic apolipoprotein which is mainly secreted by the liver as a component of triglyceride-rich lipoproteins and high density lipoproteins whose importance in plasma lipoprotein metabolism is increasingly evident. At present, the only way to obtain native ApoC-I is separating it from human plasma. The methods have some restrictions on source, the complicated technology, the potential infections and a high cost which limits the research and application of native ApoC-I. Because of its small size, ApoC-I has previously been prepared by peptide synthesis which is also limited by a high cost. Therefore, in this study, a Pichia pastoris expression system was first used to obtain a high level expression of secreted, recombinant human ApoC-I (rhApoC-I).     

Conformation propensities of des-acyl-ghrelin as probed by CD and NMR

Des-acyl-ghrelin is a 28 amino acid peptide secreted by both human and rat stomach. Together with ghrelin and obestatin, it is obtained by post-translational modification of a 117 aminoacid prepropeptide mainly expressed in distinct endocrine cell type in the stomach. Although its receptor has not been unambiguously identified so far, des-acyl-ghrelin is considered one of the strongest antagonists of ghrelin in activating the growth hormone secretagogue receptor (GHS-R). Here the secondary structure of des-acyl-ghrelin in different experimental conditions has been investigated and compared with that of obestatin, a bioactive peptide having similar biological functions. CD and NMR techniques have been combined for gaining the desired conformational features. The obtained structures support a steady alpha-helix structure spanning residues from 7 to 14, very similar to that observed for obestatin at the same experimental conditions, leading to suggest that a similar secondary structure can be associated with the similar biological role.     

Solution structure of alpha-conotoxin ImI determined by two-dimensional NMR spectroscopy.

The three-dimensional structure of alpha-conotoxin ImI, a potent antagonist targeting the neuronal alpha7 subtype of nicotinic acetylcholine receptor (nAChR), has been investigated by NMR spectroscopy. On the basis of 181 experimental constraints, a total of 25 converged structures were obtained. The average pairwise atomic root mean square difference is 0.40+/-0.11 A for the backbone atoms. The resulting structure indicates the presence of two successive type I beta-turns and a 310 helix for residues Cys2-Cys8 and Ala9-Arg11, respectively, and shows a significant structural similarity to that of alpha-conotoxin PnIA, which is also selective for the neuronal nAChR.     

Conformational behavior of fragments of adrenocorticotropin and their antisense peptides determined by NMR spectroscopy and CD spectropolarimetry

An ‘antisense’ peptide (‘HTCA’), whose sequence was generated by reading the antisense RNA sequence corresponding to ACTH(1–24) was shown to bind ACTH(1–24) with a K_d of 0.3 nM in a solid-matrix binding assay [(1986) Biochem. J. 234, 679–683]. Two-dimensional NMR spectra were used to examine the conformational behavior in methanol and in water solution of two fragments of adrenocorticotropin, ACTH(1–24) and ACTH(1–13), as well as their antisense peptides, HTCA and HTCA(12–24). The conformations are extended chains in these solutions, both as isolated molecules and when mixed with their antisense complements. The K_d values are greater than 1 mM.     

Secondary structure of human apolipoprotein A-I(1-186) in lipid-mimetic solution

The solution structure of an apoA-I deletion mutant, apoA-I(1-186) was determined by the chemical shift index (CSI) method and the torsion angle likelihood obtained from shift and sequence similarity (TALOS) method, using heteronuclear multidimensional NMR spectra of [u-(13)C, u-(15)N, u-50% (2)H]apoA-I(1-186) in the presence of sodium dodecyl sulfate (SDS). The backbone resonances were assigned from a combination of triple-resonance data (HNCO, HNCA, HN(CO)CA, HN(CA)CO and HN(COCA)HA), and intraresidue and sequential NOEs (three-dimensional (3D) and four-dimensional (4D) 13C- and 15N-edited NOESY). Analysis of the NOEs, H(alpha), C(alpha) and C' chemical shifts shows that apoA-I(1-186) in lipid-mimetic solution is composed of alpha-helices (which include the residues 8-32, 45-64, 67-77, 83-87, 90-97, 100-140, 146-162, and 166-181), interrupted by short irregular segments. There is one relatively long, irregular and mostly flexible region (residues 33-44), that separates the N-terminal domain (residues 1-32) from the main body of protein. In addition, we report, for the first time, the structure of the N-terminal domain of apoA-I in a lipid-mimetic environment. Its structure (alpha-helix 8-32 and flexible linker 33-44) would suggest that this domain is structurally, and possibly functionally, separated from the other part of the molecule.     

Conformation of the second disulfide loop in human transforming growth factor-alpha studied by two-dimensional NMR spectroscopy

The determination of the conformation of a cyclic heptadecapeptide derived from the second loop of human transforming growth factor-α, [Ala²¹]-hTGF-α-(16–32), is described. Two-dimensional homonuclear Hartmann–Hahn and rotating-frame cross-relaxation spectroscopy in H₂O were used to obtain the complete proton resonance assignments and the necessary distance constraints between nonbonded hydrogen atoms to derive a conformation without involving any energy minimization. The result is an ellipsoidal-shaped structure with a turn at Gly19 and a bend formed by residues 26–29, Gln-Glu-Asp-Lys. Comparison is made with the second loop of human epidermal growth factor and the results are discussed in terms of receptor binding and biological activity.     

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Three-dimensional solution structure of apo-neocarzinostatin from Streptomyces carzinostaticus determined by NMR spectroscopy.

The three-dimensional solution structure of apo-neocarzinostatin has been resolved from nuclear magnetic resonance spectroscopy data. Up to 1034 constraints were used to generate an initial set of 45 structures using a distance geometry algorithm (DSPACE). From this set, ten structures were subjected to refinement by restrained energy minimization and molecular dynamics. The average atomic root mean square deviations between the final ten structures and the mean structure obtained by averaging their coordinates run from 0.085 nm for the best defined beta-sheet regions of the protein to 0.227 nm for the side chains of the most flexible loops. The solution structure of apo-neocarzinostatin is closely similar to that of the related proteins, macromomycin and actinoxanthin. It contains a seven-stranded antiparallel beta-barrel which forms, together with two external loops, a deep cavity that is the chromophore binding site. It is noteworthy that aromatic side chains extend into the binding cleft. They may be responsible for the stabilization of the holo-protein complex and for the chromophore specificity within the antitumoral family.     

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy

The broad spectrum of antibacterial activities of host defense cationic antimicrobial peptides (AMPs) arises from their ability to perturb membrane integrity of the microbes. The mechanisms are often thought to require assembly of AMPs on the membrane surface to form pores. However, three dimensional structures in the oligomeric form of AMPs in the context of lipid membranes are largely limited. Here, we demonstrate that a 22-residue antimicrobial peptide, termed VK22, derived from fowlicidin-1, a cathelicidin family of AMP from chicken oligomerizes into a predominantly tetrameric state in zwitterionic dodecylphosphocholine (DPC) micelles. An ensemble of NMR structures of VK22 determined in 200mM perdeuterated DPC, from 755 NOE constrains including 19 inter-helical NOEs, had revealed an assembly of four helices arranged in anti-parallel fashion. Hydrogen bonds, C(α)H-O=C types, and van der Waals interactions among the helical sub-units appear to be involved in the stabilization of the quaternary structures. The central region of the barrel shaped tetrameric bundle is non-polar with clusters of aromatic residues, whereas all the cationic residues are positioned at the termini. Paramagnetic spin labeled NMR experiments indicated that the tetrameric structure is embedded into micelles such that the non-polar region located inside the lipid acyl chains. Structure and micelle localization of a monomeric version, obtained from substitution of two Tyr residues with Ala, of the peptide is also compared. The mutated peptide VK22AA has been found be localized at the surface of the micelles. The tetrameric structure of VK22 delineates a small water pore that can be larger in the higher order oligomers. As these results provide structural insights, at atomic resolution, into the oligomeric states of a helical AMP in lipid environment, the structural details may be further utilized for the design of novel self-assembled membrane protein mimics.     

Conformation of the ATP binding peptide in actin revealed by proton NMR spectroscopy

The actin peptide 106-124 exists in a completely conserved region of the sequence and binds strongly to both ATP and tripolyphosphate. Binding particularly affects residues 116 and 118 and generally affects the two segments 115-118 and 121-124 [Barden, J. A., & Kemp, B. E. (1987) Biochemistry 26, 1471-1478]. One-dimensional nuclear Overhauser enhancement difference spectroscopy was used to detect molecular interactions between both adjacent and nonadjacent residues. The N-terminal segment 106-112 was found to be largely extended. A sharp bend was detected between Pro-112 and Lys-113. The triphosphate moiety binds to the strongly hydrophilic central segment of the peptide. Evidence was obtained for a reverse turn involving residues 121-124. Amide proton temperature coefficients and coupling constants provide evidence for a type I beta-turn. A model of the ATP binding site is proposed together with its relationship to other parts of the actin structure and to the phalloidin binding site.