Conformational analysis of the Sda determinant-containing tetrasaccharide and two mimics in aqueous solution by using 1H NMR ROESY spectroscopy in combination with MD simulations

The conformational behaviour of the spacer-linked synthetic Sd^a tetrasaccharide -d-GalpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (1) and the two mimics -d-Galp-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (2) and -d-GlcpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (3) were investigated by ¹H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. Experimental 2D ¹H ROESY cross-peak intensities (ROEs) of the tetrasaccharides were compared with calculated ROEs derived from MD trajectories using the CROSREL program. Analysis of these data indicated that the oligosaccharidic skeletons of the compounds 1–3 are rather rigid, especially the -d-Hex(NAc)-(14)-[-Neu5Ac-(23)]--d-Galp fragments. The - Neu5-Ac-(23)--d-Galp linkage occurred in two different energy minima in the three-dimensional structure of the compounds 1–3 in aqueous solution. Experimental data and dynamics simulations supported the finding that the higher energy rotamer (CHEAT forcefield) was abundant in compounds 1 and 3 due to the existence of a hydrogen bond between the carboxyl group of the sialic acid and the acetamido group of the terminal monosaccharide (GalNAc or GlcNAc) unit. The conformational similarity between 1 and 3 leads to the suggestion that also their activities will be alike.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Solution conformation and dynamics of a tetrasaccharide related to the LewisX antigen deduced by NMR relaxation measurements

¹H-NMR cross-relaxation rates and nonselectivelongitudinal relaxation times have been obtained at two magnetic fields (7.0and 11.8 T) and at a variety of temperatures for the branchedtetrasaccharide methyl3-O--N-acetyl-galactosaminyl--galactopyranosyl-(14)[3-O--fucosyl]-glucopyranoside (1), an inhibitor of astrocyte growth. Inaddition, ¹³C-NMR relaxation data have also been recorded atboth fields. The ¹H-NMR relaxation data have been interpretedusing different motional models to obtain proton–proton correlationtimes. The results indicate that the GalNAc and Fuc rings display moreextensive local motion than the two inner Glc and Gal moieties, since thosepresent significantly shorter local correlation times. The¹³C-NMR relaxation parameters have been interpreted in termsof the Lipari–Szabo model-free approach. Thus, order parameters andinternal motion correlation times have been deduced. As obtained for the¹H-NMR relaxation data, the two outer residues possess smallerorder parameters than the two inner rings. Internal correlation times are inthe order of 100 ps. The hydroxymethyl groups have also different behaviour,with the exocyclic carbon on the glucopyranoside unit showing the highestS². Molecular dynamics simulations using a solvated systemhave also been performed and internal motion correlation functions have beendeduced from these calculations. Order parameters and interproton distanceshave been compared to those inferred from the NMR measurements. The obtainedresults are in fair agreement with the experimental data.     

Two-dimensional rotating-frame Overhauser spectroscopy (ROESY) and (13)C NMR study of the interactions between maltodextrin and an anionic surfactant

Rotational frame nuclear Overhauser effect spectroscopy (ROESY) and (13)C NMR measurements were carried out to study the molecular interaction between maltodextrin, a digestive byproduct of starch, and an anionic surfactant. Significant differences in chemical shifts were observed when sodium dodecyl sulfate (SDS) was introduced into the maltodextrin (DE 10) solutions. (13)C NMR measurement indicated that there were downfield shifts and broadening of peaks, especially in the region of 75-81 and 100-103 ppm, which were assigned to carbons 1 and 4 of the d-glucopyranose residues of maltodextrin, respectively. ROESY spectra indicated cross-peaks between the SDS and maltodextrin protons. These peaks can arise only in the case of the designated SDS protons and maltodextrin protons being less than 0.5 nm apart for a substantial period of time. The most intense cross-peaks are those between the central CH(2) protons of SDS near 1.2 ppm and the maltodextrin protons ranging from 3.5 to 3.9 ppm. The SDS-H3 CH(2) protons were resolved from the bulk of the SDS protons, with peaks and shoulders at 1.25 ppm, which indicated an especially strong interaction of the SDS hydrophobic tail with MD6 and some less intense interactions with MD2, 4, and 5.     

Dynamics of sialyl Lewis(a) in aqueous solution and prediction of the structure of the sialyl Lewis(a)-SelectinE complex

In this article we investigate all possible three-dimensional structures for sialyl Lewis^a (SLe^a) in aqueous solution and we predict without a priori experimental information its conformation when bound to SelectinE by using a combination of long molecular dynamics (MD) simulations. Based on 10 ns MD studies, three structures differing in glycosidic conformations are proposed for SLe^a in aqueous solution. Based on a 4 ns MD study of the SLe^a-SelectinE complex with initial structures derived from our prediction tools, we find that, fucose and N-acetyl neuraminic acid are in close contact with SelectinE and therefore expect interactions of the protein with these two sugar rings to be significantly more important than in the case of galactose and N-acetyl glucosamine. Our predictions indicate that the N-acetyl glucosamine of SLe^a is positioned primarily in the aqueous phase. In order to be able to interact with SLe^a the side chains of amino acid residues Lys99 and Lys111 in SelectinE appear to undergo large conformational changes when contrasted with the positions of these residues in the X-ray crystal structure. Furthermore, amino acid residues Arg97, Glu98 and Lys99 are acting as a holding arm to position the NeuNAc of SLe^a in the binding pocket.     

Synthesis of 3-C-(6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-mannopyranosyl)-1-propene: a caveat

During the preparation of 3-C-(6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-mannopyranosyl)-1-propene, using a published Sakurai-type reaction on the parent methyl glycoside, some observations were made on the sensitivity to reaction conditions that were not previously reported. This Note presents the study of this allylation reaction followed by acetolysis, which ultimately led to the best conditions to obtain the C-glycoside, and on further transformations to yield the corresponding aldehydic and acidic derivatives.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Solution structure and mapping of a very weak calcium-binding site of human translationally controlled tumor protein by NMR

Human translationally controlled tumor protein (TCTP) is a growth-related, calcium-binding protein. We determined the solution structure and backbone dynamics of human TCTP, and identified the calcium-binding site of human TCTP using multi-dimensional NMR spectroscopy. The overall structure of human TCTP has a rather rigid well-folded core and a very flexible long loop connected by a short two-strand β-sheet, which shows a conserved fold in the TCTP family. The C-terminal portions of loop L_α3β8 and strand β9 and the N-terminal region of strand β8 may form a calcium-binding site in the human TCTP structure, which is largely conserved in the sequence alignment of TCTPs. The K_d value for the calcium binding is 0.022-0.025 M indicating a very weak calcium-binding site.     

NMR solution structure of BmK-betaIT, an excitatory scorpion beta-toxin without a 'hot spot' at the relevant position

BmK-betaIT (previously named as Bm32-VI in the literature), an excitatory scorpion beta-toxin, is purified from the venom of the Chinese scorpion Buthus martensii Karsch. It features a primary sequence typical of the excitatory anti-insect toxins: two contiguous Cys residues (Cys37-Cys38) and a shifted location of the fourth disulfide bridges (Cys38-Cys64), and demonstrates bioactivity characteristic of the excitatory beta-toxins. However, it is noteworthy that BmK-betaIT is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is the first example having a non-glutamate residue at the relevant position in the excitatory scorpion beta-toxin subfamily. The 3D structure of BmK-betaIT is determined with 2D NMR spectroscopy and molecular modeling. The solution structure of BmK-betaIT is closely similar to those of BmK IT-AP and Bj-xtrIT, only distinct from the latter by lack of an alpha(0)-helix. The surface functional patch comparison with those of BmK IT-AP and Bj-xtrIT reveals their striking similarity in the spatial arrangement. These results infer that the functional surface of beta-toxins is composed of two binding regions and a functional site. The main binding site is consisted of hydrophobic residues surrounding the alpha(1)-helix and its preceding loop, which is common to all beta-type scorpion toxins affecting Na(+) channels. The second binding site, which determines the specificity of the toxin, locates at the C-terminus for excitatory insect beta-toxin, while rests at the beta-sheet and its linking loop for anti-mammal toxins. The functional site involved in the voltage sensor-trapping model, which characterizes the function of all beta-toxins, is the negatively charged residue Glu15.     

Solution conformation of endothelin, a potent vaso-constricting bicyclic peptide. A combined use of 1H NMR spectroscopy and distance geometry calculations

The solution structure of endothelin-1, a newly discovered potent bicyclic peptide vaso-constrictor agent, has been investigated using 1H NMR conformational constraints and distance geometry calculations. The conformation is constrained by two disulphide bridges between Cys1-Cys15 and Cys3-Cys11 but the NMR data and computed conformers show additional helical structure between residues Leu6 and Cys11. Our results are compared with previous conflicting reports on the solution conformation of this peptide.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Solution structure of human calcitonin gene-related peptide by 1H NMR and distance geometry with restrained molecular dynamics

The structure of human calcitonin gene-related peptide 1 (hCGRP-1) has been determined by 1H NMR in a mixed-solvent system of 50% trifluoroethanol/50% H2O at pH 3.7 and 27 degrees C. Complete resonance assignment was achieved by using two-dimensional methods. Distance restraints for structure calculations were obtained by semiquantitative analysis of intra- and interresidue nuclear Overhauser effects; in addition, stereospecific or X1 rotamer assignments were obtained for certain side chains. Structures were generated from the distance restraints by distance geometry, followed by refinement using molecular dynamics, and were compared with experimental NH-C alpha H coupling constants and amide hydrogen exchange data. The structure of hCGRP-1 in this solvent comprises an amino-terminal disulfide-bonded loop (residues 2-7) leading into a well-defined alpha-helix between residues 8 and 18; thereafter, the structure is predominantly disordered, although there are indications of a preference for a turn-type conformation between residues 19 and 21. Comparison of spectra for the homologous hCGRP-2 with those of hCGRP-1 indicates that the conformations of these two forms are essentially identical.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

Solution structure of a common substrate mimetic of cyclooxygenase-downstream synthases bound to an engineered thromboxane A2 synthase using a high-resolution NMR technique

Understanding the docking mechanism of the common substrate, prostaglandin H(2) (PGH(2)), into the active sites of different cyclooxygenase(COX)-downstream synthases is a key step toward uncovering the molecular basis of the isomerization of PGH(2) to different prostanoids. A high-resolution NMR spectroscopy was used to determine the conformational changes and solution 3D structure of U44069, a PGH(2) analogue, bound to one of the COX-downstream synthases-an engineered thromboxane A(2) synthase (TXAS). The dynamic binding was clearly observed by (1)D NMR titration. The detailed conformational change and 3D structure of U44069 bound to the TXAS were demonstrated by 2D (1)H NMR experiments using transferred NOEs. Through the assignments for the 2D (1)H NMR spectra, TOCSY, DQF-COSY, NOESY, and the structural calculations based on the NOE constraints, they demonstrated that the widely open conformation with a triangle shape of the free U44069 changed to a compact structure with an oval shape when bound to the TXAS. The putative substrate-binding pocket of the TXAS model fits the conformation of the TXAS-bound U44069 appropriately, but could not fit the free form of U44069. It was the first to provide structural information for the dynamic docking of the PGH(2) mimic of the TXAS in solution, and to imply that PGH(2) undergoes conformational changes when bound to different COX-downstream synthases, which may play important roles in the isomerization of PGH(2) to different prostanoids. The NMR technique can be used as a powerful tool to determine the conformations of PGH(2) bound to other COX-downstream synthases.     

Quantitative determination of metabolites in human urine by <Superscript>1</Superscript>H NMR spectroscopy

Conditions for registration of urinary ¹H NMR spectra have been optimized in order to achieve maximal accuracy of quantitative analysis. Urinary samples from patients with acute pancreatitis have been investigated and spectral data of identified urinary metabolites and results of their quantitative determination are given. Employment of ¹H NMR spectra is perspective for the development of new laboratory diagnostic methods.     

Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

Summary Biosynthetically directed fractional incorporation of¹³C into proteins results in nonrandom¹³C-labeling patterns that can be investigated by analysis of the¹³C–¹³C scalar coupling fine structures in heteronuclear¹³C–¹H or homonuclear¹³C–¹³C correlation experiments. Previously this approach was used for obtaining stereospecific¹H and¹³C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the¹H spin-system identifications. Studies of the hydrolysates of fractionally¹³C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of¹³C–¹³C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear¹H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the - and -methyl groups of Ile. In addition to the methyl resonances, the CH₂ groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the -carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.Abbreviations NOE nuclear Overhauser effect - fractional¹³C labeling biosynthetically directed fractional¹³C-labeling - TOCSY total correlation spectroscopy - ROESY rotating frame Overhauser enhancement spectroscopy - [¹³C,¹H]-COSY two-dimensional¹³C–¹H correlation spectroscopy - isotopomer isotope isomer - P22 c2 repressor c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues - P22 c2(1-76) N-terminal domain of the P22 c2 repressor with residues 1–76     

Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

Summary A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYIN-LITRQRY-NH₂, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15°C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined -helix in the centre, with a few residues at the termini having less well defined conformations. The spinlattice and spin-spin relaxation rates of -carbons have been determined on ¹³C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C-H vectors in the -helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.Supplementary material available from the authors: Two data tables and 10 PDB coordinate files of the calculated NMR structures of P7. One data table contains all detected and integrated NOE intensities; the other connects each proton and pseudoatom to an atom number used in the NOE table. The table contents served as input data files for CALIBA.Currently on leave from the Institute of Chemical Physics and Biophysics, Tallinn, Estonia.     

Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy.

The solution secondary structure of calcium-saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium-saturated NTnC (residues 1-90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium-saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium-saturated TnC with the X-ray crystal structure of half-saturated TnC reveals differences in the phi/psi angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular phi/psi angles in the crystal structure, producing a kink in the B helix, whereas in calcium-saturated TnC, Glu 41 has helical phi/psi angles, resulting in a straight B helix. The linker between the N and C domains of calcium-saturated TnC is flexible in the solution structure.     

Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance (13)C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the [(1)H]-(13)C NOE were determined in this study. The C alpha H relaxation measurements were compared to the previously measured (15)N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the chi(1) dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than +/-25 degrees.