Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy

The three-dimensional solution structure of ascidian trypsin inhibitor (ATI), a 55 amino acid residue protein with four disulfide bridges, was determined by means of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The resulting structure of ATI was characterized by an alpha-helical conformation in residues 35-42 and a three-stranded antiparallel beta-sheet in residues 22-26, 29-32, and 48-50. The presence of an alpha-helical conformation was predicted from the consensus sequences of the cystine-stabilized alpha-helical (CSH) motif, which is characterized by an alpha-helix structure in the Cys-X(1)-X(2)-X(3)-Cys portion (corresponding to residues 37-41), linking to the Cys-X-Cys portion (corresponding to residues 12-14) folded in an extended structure. The secondary structure and the overall folding of the main chain of ATI were very similar to those of the Kazal-type inhibitors, such as Japanese quail ovomucoid third domain (OMJPQ3) and leech-derived tryptase inhibitor form C (LDTI-C), although ATI does not show extensive sequence homology to these inhibitors except for a few amino acid residues and six of eight half-cystines. On the basis of these findings, we realign the amino acid sequences of representative Kazal-type inhibitors including ATI and discuss the unique structure of ATI with four disulfide bridges.     

Solution structure of apamin determined by nuclear magnetic resonance and distance geometry

The solution structure of the bee venom neurotoxin apamin has been determined with a distance geometry program using distance constraints derived from NMR. Twenty embedded structures were generated and refined by using the program DSPACE. After error minimization using both conjugate gradient and dynamics algorithms, six structures had very low residual error. Comparisons of these show that the backbone of the peptide is quite well-defined with the largest rms difference between backbone atoms in these structures of 1.34 A. The side chains have far fewer constraints and show greater variability in their positions. The structure derived here is generally consistent with the qualitative model previously described, with most differences occurring in the loop between the beta-turn (residues 2-5) and the C-terminal alpha-helix (residues 9-17). Comparisons are made with previously derived models from NMR data and other methods.     

Solution structure of neuromedin B by (1)H nuclear magnetic resonance spectroscopy.

The solution structure of neuromedin B (NMB) was investigated using two-dimensional nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking environments. NMB adopts a relaxed helical conformation from Trp(4) to Met(10) in 50% aqueous 2,2, 2-trifluoroethanol (TFE) solution and in 150 mM SDS micelles. Sidechain atoms of the three residues, Trp(4), His(8) and Phe(9) orient toward the same direction and these residues might play a key role on interacting with hydrophobic acyl chains of the phospholipids in the membrane. NOESY experiments performed on NMB in non-deuterated SDS micelle show that aromatic ring protons of Trp(4) and Phe(9) residues are in close contact with methylene protons of SDS micelles. In addition, proton longitudinal relaxation data proved that the interactions between NMB with SDS micelle are characterized as extrinsic interaction. Trp(4) and Phe(9) seem to be important in interaction with receptor and this agrees with the previous studies of structure-activity relationship (Howell, D.C. et al. (1996) Int. J. Pept. Protein Res. 48, 522-531). These conformational features might be helpful in understanding the molecular mechanism of the function of NMB and developing the efficient drugs.     

Assignment and secondary structure of calcium-bound human S100B

The NMR assignments of backbone ¹H, ¹³C,and ¹⁵N resonances for calcium-bound human S100B werecompleted via heteronuclear multidimensional NMR spectroscopic techniques.NOE correlations, amide exchange, ³J_HN_Hcoupling constants, and CSI analysis were used to identify the secondarystructure for Ca-S100B. The protein is comprised of four helices (helix I,Glu²-;Arg²⁰; helix II,Glu³¹-;Asn³⁸; helix III,Gln⁵⁰-;Thr⁵⁹; helix IV,Phe⁷⁰-;Phe⁸⁷), three loops (loop I,Glu²¹-;His²⁵; loop II,Glu³⁹-;Glu⁴⁹; loop III,Leu⁶⁰-;Gly⁶⁶), and two -strands(strand I, Lys²⁶>-;Lys²⁸; strand II,Glu⁶⁷-;Asp⁶⁹) which form a shortantiparallel -sheet. Helix IV is extended by approximately one turnwhen compared to the secondary structures of apo-rat [Drohat et al. (1996)Biochemistry, 35, 11577-;11588] and bovine S100B [Kilby et al. (1996)Structure, 4, 1041-;1052]. In addition, several residues outside thecalcium-binding loops in S100B undergo significant backbone chemical shiftchanges upon binding calcium which are not observed in the related proteincalbindin D_9k. Together these observations support previoussite-directed mutagenesis, absorption spectroscopy, and cysteine chemicalreactivity experiments, suggesting that the C-terminus in Ca-S100B isimportant for interactions with other proteins.     

Solution structure of tertiapin determined using nuclear magnetic resonance and distance geometry

The solution structure of tertiapin, a 21-residue bee venom peptide, has been characterized by circular dichroism (CD), two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and distance geometry. A total of 21 lowest error structures were obtained from distance geometry calculations. Superimposition of these structures shows that the backbone of tertiapin is very well defined. One type-I reverse turn from residue 4 to 7 and an α-helix from residue 12 to 19 exist in the structure of tertiapin. The α-helical region is best defined from both conformational analysis and structural superimposition. The overall three-dimensional structure of tertiapin is highly compact resulting from side chain interactions. The structural information obtained from CD and NMR are compared for both tertiapin and apamin (ref. 3), another bee venom peptide. Tertiapin and apamin have some similar secondary structure, but display different tertiary structures. © 1993 Wiley-Liss, Inc.     

Solution structure of human Mts1 (S100A4) as determined by NMR spectroscopy

Mts1 is a member of the S100 family of Ca2+-binding proteins and is implicated in promoting tumor progression and metastasis. To better understand the structure-function relationships of this protein and to begin characterizing its Ca2+-dependent interaction with protein binding targets, the three-dimensional structure of mts1 was determined in the apo state by NMR spectroscopy. As with other S100 protein family members, mts1 is a symmetric homodimer held together by noncovalent interactions between two helices from each subunit (helices 1, 4, 1', and 4') to form an X-type four-helix bundle. Each subunit of mts1 has two EF-hand Ca2+-binding domains: a pseudo-EF-hand (or S100-hand) and a typical EF-hand that are brought into proximity by a small two-stranded antiparallel beta-sheet. The S100-hand is formed by helices 1 and 2, and is similar in conformation to other members of the S100 family. In the typical EF-hand, the position of helix 3 is similar to that of another member of the S100 protein family, calcyclin (S100A6), and less like that of other S100 family members for which three-dimensional structures are available in the calcium-free state (e.g., S100B and S100A1). The differences in the position of helix 3 in the apo state of these four S100 proteins are likely due to variations in the amino acid sequence in the C-terminus of helix 4 and in loop 2 (the hinge region) and could potentially be used to subclassify the S100 protein family.     

Solution conformation of conotoxin GI determined by 1H nuclear magnetic resonance spectroscopy and distance geometry calculations

Conformational analysis of conotoxin GI, one of the neurotoxic peptides produced by a marine snail, genus Conus, was performed by a combination of nuclear magnetic resonance spectroscopy (NMR) and distance geometry calculations. The resulting conformers on minimization of the target function were classified into two groups. The difference in the structures of the conformers is mainly due to the difference in the orientation of the side chain of the tyrosyl residue. The results show that the solution structure of conotoxin GI satisfies the conformational requirements for the biological activity of an antagonist toward nicotinic cholinergic receptors elucidated in a series of studies on alkaloids. The structure is discussed on the basis of the results of comparison of the atomic arrangements of the active sites of snake venom peptides and molecular models based on the results of secondary structure prediction.     

Solution and ion-complexed conformations of beauvericin determined by proton magnetic resonance spectroscopy

The conformational properties of the cyclohexadepsipeptide antibiotic Beauvericin have been investigated by 1H-NMR spectroscopy in polar (C2H3O2H) and non-polar (CCl4, C62H6, C2HCl3) solvents and in two solvent mixtures; one a mixture of a polar and non-polar solvent (C2H3O2H/CCl4) and the other an aromatic solvent in a non-polar environment (C62H6/CCl4). The ion-complexation properties of Beauvericin with alkali metal halides (Li+, Na+, K+, Cs+) have also been studied. It is demonstrated that changes in chemical shifts of Beauvericin with concentration, with polarity of solvent or with added alkali metal ion reflect changes not only in the solvent properties but also changes in backbone conformation and changes due to ion-complexation, where appropriate, and therefore cannot be used, by themselves, to determine the conformation of the molecule, its self-aggregation properties, or the stoichiometry of the metal ion-complex. The backbone conformations of Beauvericin in different environments are determined by methods that are independent of chemical shift analysis; i.e., by measurements of 5J(HH) magnitudes observed between the alpha-CH protons of the L-phenylalanine and D-hydroxyisovaleric acid (DHyIv) residues and by nuclear Overhauser effect measurements observed between alpha-CH(HyIv) and (N)-CH3(Phe) proton signals. In the knowledge of these results the chemical shifts of Beauvericin in different environments can then be rationalised. It is found that the conformation of Beauvericin in a polar solvent is different from that found in a non-polar solvent and from that found for the in the ion-complexed form is similar to that found in non-polar solvents. By taking into account the conformational properties of the L-phenylalanine and DHyIv side-chains, it is possible to assign unambiguously the magnetically non-equivalent beta-CH2(Phe) and gamma Me(HyIv) proton signals and so elucidate the complete conformational behaviour of the uncomplexed forms of Beauvericin in a polar and a non-polar environment, and of the ion-complexed form of Beauvericin in a polar solvent.     

Solution conformation of endothelin determined by nuclear magnetic resonance and distance geometry

The solution conformation of endothelium-derived vasoconstrictor peptide, endothelin, has been determined by two-dimensional 1H-NMR spectroscopy and distance geometry. Conformation in the N-terminal core region (residues 1-15) is well-defined and a characteristic is the helix-like conformation in the segment from Lys9 to Cys15. Contrarily, the C-terminal tail region (residues 16-21) does not assume a defined conformation and there are no specific interactions between the core and the tail regions.     

Three-dimensional structure of an alpha-amylase inhibitor HAIM as determined by nuclear magnetic resonance methods

The three-dimensional structure of an alpha-amylase inhibitor, HAIM, composed of 78 amino acids, was analyzed by two-dimensional NMR techniques. Sequence-specific assignments were made for the amino acid residues from Ile-6 to Cys-72. Distance geometry analysis of the interresidue NOEs revealed that the HAIM molecule consists of two beta-sheets, as is the case in a homologous alpha-amylase inhibitor, Tendamistat, though one of its beta-strands is much shorter than that of Tendamistat. The combination of molecular modeling from Tendamistat and distance geometry analysis was confirmed to be useful for our purpose.     

Elucidation of glycolipid structure by proton nuclear magnetic resonance spectroscopy

The primary structure of the oligosaccharide moiety of a glycosphingolipid can be elucidated by employing high-field proton nuclear magnetic resonance (NMR) spectroscopy. Information with respect to the composition and configuration of its sugar residues, and the sequence and linkage sites of the oligosaccharide chain can be obtained by employing a variety of one- and two-dimensional techniques. The latter include both scalar and dipolar correlated two-dimensional NMR spectroscopy. These techniques are also useful in establishing the solution conformation (secondary structure) of the oligosaccharide moiety. Examples in utilizing these techniques in elucidating the primary and secondary structures of glycolipids are presented.     

Three-dimensional solution structure of the reduced form of Escherichia coli thioredoxin determined by nuclear magnetic resonance spectroscopy

The three-dimensional solution structure of reduced (dithiol) thioredoxin from Escherichia coli has been determined with distance and dihedral angle constraints obtained from 1H NMR spectroscopy. Reduced thioredoxin has a well-defined global fold consisting of a central five-strand beta-sheet and three long helices. The beta-strands are packed in the sheet in the order beta 1 beta 3 beta 2 beta 4 beta 5, with beta 1, beta 3, and beta 2 parallel and beta 2, beta 4, and beta 5 arranged in an antiparallel fashion. Two of the helices connect strands of the beta-sheet: alpha 1 between beta 1 and beta 2 and alpha 2 between beta 2 and beta 3. Strands beta 4 and beta 5 are connected by a short loop that contains a beta-bulge. Strands beta 3 and beta 4 are connected by a long loop that contains a series of turn-like or 3(10) helical structures. The active site Cys-Gly-Pro-Cys sequence forms a protruding loop between strand beta 2 and helix alpha 2. The structure is very similar overall to that of oxidized (disulfide) thioredoxin obtained from X-ray crystal structure analysis but differs in the local conformation of the active site loop. The distance between the sulfurs of Cys 32 and Cys 35 increases from 2.05 A in the disulfide bridge to 6.8 +/- 0.6 A in the dithiol of reduced thioredoxin, as a result of a rotation of the side chain of Cys 35 and a significant change in the position of Pro 34. This conformational change has important implications for the mechanism of thioredoxin as a protein disulfide oxidoreductase.     

Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin. SCM is more stable than the native monellin for both heat and acidic environments. Data from gel filtration HPLC and NMR indicate that the SCM exists as a monomer in aqueous solution. The solution structure of SCM has been determined by nuclear magnetic resonance (NMR) spectroscopy and dynamical simulated annealing calculations. A stable alpha-helix spanning residues Phe11-Ile26 and an antiparallel beta-sheet formed by residues 2-5, 36-38, 41-47, 54-64, 69-75, and 83-88 have been identified. The sheet was well defined by backbone-backbone NOEs, and the corresponding beta-strands were further confirmed by hydrogen bond networks based on amide hydrogen exchange data. Strands beta2 and beta3 are connected by a small bulge comprising residues Ile38-Cys41. A total of 993 distance and 56 dihedral angle restraints were used for simulated annealing calculations. The final simulated annealing structures (k) converged well with a root-mean-square deviation (rmsd) between backbone atoms of 0.49 A for secondary structural regions and 0.70 A for backbone atoms excluding two loop regions. The average restraint energy-minimized (REM) structure exhibited root-mean-square deviations of 1.19 A for backbone atoms and 0.85 A for backbone atoms excluding two loop regions with respect to 20 k structures. The solution structure of SCM revealed that the long alpha-helix was folded into the concave side of a six-stranded antiparallel beta-sheet. The side chains of Tyr63 and Asp66 which are common to all sweet peptides showed an opposite orientation relative to H1 helix, and they were all solvent-exposed. Residues at the proposed dimeric interface in the X-ray structure were observed to be mostly solvent-exposed and demonstrated high degrees of flexibility.     

Rate constants determined by nuclear magnetic resonance

Fast kinetic methods are used to measure reactions that take place in less time than required to mix the reagents manually and to measure the reaction by usual methods, like UV-visible spectrophotometry and fluorescence. The best known of them are rapid-mixing and relaxation methods, which are used for reactions with half-times in the millisecond and microsecond ranges, respectively. The picosecond range is usually measured with electrical field and ultrasonic waves (A. Cornish-Bowden, 1976, Principles of Enzyme Kinetics, pp. 164-167, Butterworths, London). Normally these very fast rates occur when a ligand binds to or dissociates from a protein. When the binding is mediated only by the diffusion, the lower limit of the association rate constant (k(on)) should not exceed the value of a diffusion-controlled reaction (around 10(10) M(-1) s(-1)). Therefore, the values most frequently found for these rate constants, for example, in the association of a substrate with an enzyme, are in the range 10(6) to 10(9) M(-1) s(-1) (M. Eigen and G. G. Hammes, 1963, Adv. Enzymol. 25, 1-38). The values for the dissociation rate constants (k(off)) for these reactions, which depend on the equilibrium constant for the enzyme-substrate complex interaction, are in the range 10(1) to 10(5) s(-1), most often between 10(3) and 10(4) s(-1) (A. Fersht, 1999, Structure and Mechanism in Protein Science, pp. 164-165, Freeman, New York). If the equilibrium constant is known, and the value of koff is determined by nuclear magnetic resonance (NMR), as described in this chapter, the value of k(on) can be calculated; this should not exceed the value of diffusion rate in the media in which the reaction is performed.     

Solution conformation of endothelin and point mutants by nuclear magnetic resonance spectroscopy.

Two-dimensional NMR techniques were utilized to determine the secondary structural elements of endothelin-1 (ET-1), a potent vasoconstrictor peptide, and two of its point mutants, Met-7 to Ala-7 (ETM7A), and Asp-8 to Ala-8 (ETD8A) in acetic acid-d3/water solution. Sequence specific NMR assignments were determined for all three peptides, as well as chemical shifts and NOE connectivity patterns. The chemical shifts of ET-1 and ETM7A are identical (+/- 0.05 ppm) except for the site of substitution, whereas marked shift changes were detected between ET-1 and ETD8A. These chemical shift differences imply that the Asp-8 to Ala-8 mutation has induced a conformational change relative to the parent conformation. All three molecules show the same basic nuclear Overhauser effect (NOE) pattern, which suggests that the gross conformation of all three molecules is the same. Small changes in sequential NOE intensities and changes in medium-range NOE patterns indicate that there are subtle conformational differences between ET-1 and ETD8A.     

Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution.

The 1:1 complex of the mutant Antp(C39----S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three-dimensional structure were based on the use of the fully 15N-labelled protein, two-dimensional homonuclear proton NOESY with 15N(omega 2) half-filter, and heteronuclear three-dimensional NMR experiments. Based on nearly complete sequence-specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H-1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix-turn-helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix-turn-helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39----S) homeodomain no defined spatial structure could be found for the apparently flexible N-terminal segment comprising residues 0-6.     

Lipopolysaccharide‐bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad‐spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram‐negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram‐negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α‐helical structure in a solution containing LPS. For NMR experiments, we expressed ¹⁵N‐labeled and ¹³C‐labeled CP1 in bacterial cells and successfully assigned almost all backbone and side‐chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr‐NOE) experiments in LPS. We performed ¹⁵N‐edited and ¹³C‐edited Tr‐NOE spectroscopy for CP1 bound to LPS. Tr‐NOE peaks were observed at the only C‐terminal region of CP1 in LPS. The results of structure calculation indicated that the C‐terminal region (Lys15–Gly29) formed the well‐defined α‐helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Solution structure of the antimicrobial peptide gaegurin 4 by H and 15N nuclear magnetic resonance spectroscopy.

Gaegurin 4 (GGN4) is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an alpha-helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic alpha-helices extending from residues 2-10 and from residues 16-32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix-assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C-terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.     

Metabolism and structure of triacylglycerols in rat epididymal fat pad adipocytes determined by 13C nuclear magnetic resonance

Carbon-13 nuclear magnetic resonance (NMR) methods have been applied to a study of the structure and metabolism of the triacylglycerols from rat epididymal fat pad adipocytes. Complete NMR signal assignments are provided for adipocytes, the extracted triacylglycerols, and methyl esters of the derived fatty acids. 13C NMR yielded rapid, nondestructive, quantitative analysis of the amounts of unsaturation of the fatty acyl chains; in cells from rats given ad libitum access to a standard laboratory diet the predominant fatty acids were found to be palmitate (29.9%), oleate (27.9%), and linoleate (34.1%). These results agreed with gas chromatographic separation of the derived methyl esters of the extracted lipids. Lipid dynamics were examined in situ and showed a substantial restriction of motion of glyceride-glycerol as compared with free glycerol; the nuclear magnetic spin-lattice relaxation times for free glycerol of 2.52 +/- 0.12 (C1,3) and 4.37 +/- 0.21 (C2) s decreased to 0.15 +/- 0.009 and 0.21 +/- 0.013 s, respectively, upon esterification. Segmental motion of the chains, monitored by relaxation time measurements, increased progressively from the alpha-carbon (nT1 = 0.70 s) to the methyl ends of the chains (nT1 = 9.63). The incorporation of C-13-labeled substrates ([1-13C]glucose and [3-13C]lactate) into the glycerol moiety of triacylglycerols was monitored in real time, in the presence of insulin. Lactate (10 mM) inhibited the incorporation of glucose (5.5 mM) into glyceride-glycerol. Lipolysis at the natural abundance level of 13C was measured in the presence of 10 microM isoproterenol. Simultaneous lipogenesis and lipolysis were found to occur in situ and were measured with the aid of [1-13C]glucose and isoproterenol; the labeling pattern of medium glycerol versus extracted triacylglycerols was significantly different from that found using natural abundance glucose. Our results indicate that 13C NMR is a useful new method for the real-time monitoring of lipid structure and metabolism in vivo.