Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

Residues Leu261, Trp264, and Phe265 account for apolipoprotein E-induced dyslipidemia and affect the formation of apolipoprotein E-containing high-density lipoprotein

Overexpression of apolipoprotein E (apoE) induces hypertriglyceridemia in apoE-deficient mice, which is abrogated by deletion of the carboxy-terminal segment of residues 260-299. We have used adenovirus-mediated gene transfer in apoE-/- and apoA-I-/- mice to test the effect of three sets of apoE mutations within the region of residues 261-265 on the induction of hypertriglyceridemia, the esterification of cholesterol of very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL), and the formation of spherical or discoidal apoE-containing HDL. A single-amino acid substitution (apoE4[Phe265Ala]) induced hypertriglyceridemia in apoE-/- or apoA-I-/- mice, promoted the accumulation of free cholesterol in the very low-density lipoprotein (VLDL) and HDL region, and decreased HDL cholesterol levels. A double substitution (apoE4[Leu261Ala/Trp264Ala]) induced milder hypertriglyceridemia and increased HDL cholesterol levels. A triple substitution (apoE4[Leu261Ala/Trp264Ala/Phe265Ala] or apoE2[Leu261Ala/Trp264Ala/Phe265Ala]) did not induce hypertriglyceridemia and increased greatly the HDL cholesterol levels. Electron microscopy (EM) analysis of the HDL fractions showed that apoE4[Leu261Ala/Trp264Ala/Phe265Ala] and apoE2[Leu261Ala/Trp264Ala/Phe265Ala] contained spherical HDL, apoE4[Leu261Ala/Trp264Ala] contained mostly spherical and few discoidal HDL particles, and apoE4[Phe265Ala] contained discoidal HDL. We conclude that residues Leu261, Trp264, and Phe265 play an important role in apoE-induced hypertriglyceridemia, the accumulation of free cholesterol in VLDL and HDL, and the formation of discoidal HDL. Substitution of these residues with Ala improves the apoE functions by preventing hypertriglyceridemia and promoting formation of spherical apoE-containing HDL.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or D-Tpi.

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the D forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu13 psi (CH2NH)Leu14-bombesin(6-14) and Leu13 psi(CH2NH)Phe14-bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of 125I-Tyr4-bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [3H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist D-Tpi6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3095) was slightly more potent in these assays than D-Trp6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3125). Nevertheless, D-Trp6,Leu13 psi (CH2NH)Phe14-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC50 value less than 1 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational studies of human Des-Trp1,Nle12-minigastrin in water-trifluoroethanol mixtures by 1H NMR and circular dichroism

The 1H NMR spectrum of the title peptide, H-Leu-(Glu)5-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, in 90% H2O/10% D2O was assigned by two-dimensional methods, and the displacement of the proton resonances upon addition of 2,2,2-trifluoroethanol (TFE) was followed. This permitted the assignment of the spectrum in 90% TFE/10% D2O. While the water conformation of the minigastrin analogue is random, the CD spectrum indicates that an ordered structure is present in TFE. Variable-temperature NMR data in this medium show that six amide protons have low temperature coefficients, two of the five Glu's, Trp, Nle, Asp, and Phe. These results were interpreted in terms of an alpha-helical stretch comprising the Leu and the five Glu residues and a 3(10)-helix initiated by a beta-turn at the sequence -Ala-Tyr-Gly-Trp-. Both CD and NMR data at different solvent compositions show two regions of conformational change, between 20 and 25% water and above 60% water.     

Local structural features around the C-terminal segment of Streptomyces subtilisin inhibitor studied by carbonyl carbon nuclear magnetic resonances three phenylalanyl residues

The carbonyl carbon NMR signals of the Phe residues in Streptomyces subtilisin inhibitor (SSI) were selectively observed for [F]SSI, in which all phenylalanines were uniformly labeled with [1-13C]Phe. The three enhanced resonances in the spectrum of [F]SSI were unambiguously assigned to the specific sites in the amino acid sequence by means of 15N,13C double-labeling techniques. Namely, the resonances at 174.9 and 172.6 ppm (in D2O, pH 7.3, 50 degrees C) showed the satellite peaks due to 13C-15N spin coupling in the spectra of [F,GS]SSI and [F,A]SSI, in which Ser/Gly and Ala residues were labeled with [15N]Gly/Ser and [15N]Ala, respectively, together with [1-13C]Phe. The carbonyl groups of Phe-97 and Phe-111 are involved in peptide bonds with the amino nitrogens of Ser-98 and Ala-112, respectively. These results clearly indicate that the signals at 174.5 and 172.6 ppm are due to Phe-97 and Phe-111, respectively. The signal at the lowest field (177.1 ppm) was thus assigned to the carboxyl carbon of the C-terminal Phe-113. The lifetimes of the amide hydrogens of the three Phe residues and their C-terminal-side neighbors (Ser-98 and Ala-112) were investigated by using the effect of deuterium-hydrogen exchange of amide on the line shapes (DEALS) for the Phe carbonyl carbon resonances. In this method, the NMR spectra of [F]SSI dissolved in 50% D2O (pH 7.3) were measured at various temperatures, and the line shape changes caused by deuteriation isotope shifts were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural studies of cytochrome b5: complete sequence-specific resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from pig and calf

We report complete sequence-specific proton resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from calf liver. In addition, sequence-specific resonance assignments for the main-chain amino acid protons (i.e., C alpha, C beta, and amide protons) are also reported for the porcine cytochrome b5. Assignment of the majority of the main-chain resonances was rapidly accomplished by automated procedures that used COSY and HOHAHA peak coordinates as input. Long side chain amino acid spin system identification was facilitated by long-range coherence-transfer experiments (HOHAHA). Problems with resonance overlap were resolved by examining differences between the two-dimensional 500-MHz NMR spectra of rabbit, pig, and calf proteins and by examining the temperature-dependent variation of amide proton resonances. Calculations of the aromatic ring-current shifts for protons that the X-ray crystal structure indicated were proximal to aromatic residues were found to be useful in corroborating assignments, especially those due to the large shifts induced by the heme. Assignment of NOESY cross peaks was greatly facilitated by a prediction of intensities using a complete relaxation matrix analysis based on the crystal structure. These results suggest that the single-crystal X-ray structure closely resembles that of the solution structure although there is evidence that the solution structure has a more dynamic character.     

Importance of van der Waals contact between Glu 35 and Trp 109 to the catalytic action of human lysozyme.

The importance of van der Waals contact between Glu 35 and Trp 109 to the active-site structure and the catalytic properties of human lysozyme (HL) has been investigated by site-directed mutagenesis. The X-ray analysis of mutant HLs revealed that both the replacement of Glu 35 by Asp or Ala, and the replacement of Trp 109 by Phe or Ala resulted in a significant but localized change in the active-site cleft geometry. A prominent movement of the backbone structure was detected in the region of residues 110 to 120 and in the region of residues 100 to 115 for the mutations concerning Glu 35 and Trp 109, respectively. Accompanied by the displacement of the main-chain atoms with a maximal deviation of C alpha atom position ranging from 0.7 A to 1.0 A, the mutant HLs showed a remarkable change in the catalytic properties against Micrococcus luteus cell substrate as compared with native HL. Although the replacement of Glu 35 by Ala completely abolished the lytic activity, HL-Asp 35 mutant retained a weak but a certain lytic activity, showing the possible involvement of the side-chain carboxylate group of Asp 35 in the catalytic action. The kinetic consequence derived from the replacement of Trp 109 by Phe or Ala together with the result of the structural change suggested that the structural detail of the cleft lobe composed of the residues 100 to 115 centered at Ala 108 was responsible for the turnover in the reaction of HL against the bacterial cell wall substrate. The results revealed that the van der Waals contact between Glu 35 and Trp 109 was an essential determinant in the catalytic action of HL.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Thrombin-bound conformation of the C-terminal fragments of hirudin determined by transferred nuclear Overhauser effects

The interaction of the C-terminal fragments (residues 52-65 and 55-65) of the thrombin-specific inhibitor hirudin with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution. Thrombin induces specific line broadening of the proton resonances of residues Asp(55) to Gln(65) of the synthetic hirudin fragments H-Asn-Asp-Gly-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH and acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH. This demonstrates that residues 55-65 are the predominant binding site of hirudin fragments with thrombin. Hirudin fragments take on a well-defined structure when bound to thrombin as indicated by several long-range transferred NOEs between the backbone and side-chain protons of the peptides, but they are not structured when free in solution. Particularly, transferred NOEs exist between the alpha CH proton of Glu(61) and the NH proton of Leu(64) [d alpha N(i,i+3)], between the alpha CH proton of Glu(61) and the beta CH2 protons of Leu(64) [d alpha beta(i,i+3)], and between the alpha CH proton of Glu(62) and the gamma CH2 protons of Gln(65) [d alpha gamma(i,i+3)]. These NOEs are characteristic of an alpha-helical structure involving residues Glu(61) to Gln(65). There are also NOEs between the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Distance geometry calculations suggest that in the structure of the thrombin-bound hirudin peptides all the charged residues lie on the opposite side of a hydrophobic cluster formed by the nonpolar side chains of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64).     

Importance of the C-terminal phenylalanine of gastrin for binding to the human CCK(2) receptor.

The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15,Ala17]G(11-17), [Leu15,Abu17]G(11-17), [Leu15,Val17]G(11-17), [Leu15,Leu17]G(11-17), [Leu15,Cha17]G(11-17), [Leu15,Trp17]G(11-17), [Leu15,Tic17]G(11-17), [Leu15, d-Phe17]G(11-17) and [Leu15,p-X-Phe17]G(11-17), where X = F, Cl, Br, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g+ or g- conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.     

Conformational investigation of alpha,beta-dehydropeptides. Part. IV. Beta-turn in saturated and alpha,beta-unsaturated peptides Ac-Pro-Xaa-NHCH3: NMR and IR studies.

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Val, Phe, Leu, Abu, Ala) as well as alpha,beta-unsaturated Ac-Pro-delta Xaa-NHCH3 [delta Xaa = delta Val, (Z)-delta Phe, (Z)-delta Leu, (Z)-delta Abu] were investigated in CDCl3 and CH2Cl2 by 1H-, 13C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts delta delta for NH (Xaa) and NHCH3 on going from CDCl3 to (CD3)2SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and alpha,beta-dehydropeptides (delta Xaa) on the other. Former compounds are conformationally flexible with an inverse gamma-bend, a beta-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and alpha,beta-dehydropeptides are very similar, with the type-II beta-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The beta-turn formation propensity seems to be somewhat greater in alpha,beta-unsaturated than in heterochiral peptides, but an estimation of beta-folded conformers is risky.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi.

Seven new antagonists of bombesin (Bn)/gastrin-releasing peptide (GRP) containing C-terminal Trp or Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-carboxylic acid) in a reduced peptide bond were synthesized by solid phase methods and evaluated biologically. The reduced bond in four [Leu13 psi(CH2NH)Trp14]Bn(6-14) analogs was formed by reductive alkylation at the dipeptide stage. In the case of three [Leu13 psi(CH2N)Tpi14]Bn(6-14) analogs, the Trp dipeptide with reduced bond was reacted with formaldehyde to form the corresponding Tpi derivative. These Tpi-containing analogs have a new reduced bond which is structurally more constrained. Leu13 psi(CH2N)Tpi14 analogs inhibit [125I][Tyr4]bombesin binding to Swiss 3T3 cells with IC50 values of 2-4 nM, compared to 5-10 nM for Leu13 psi(CH2NH)Trp14 analogs. Leu13 psi(CH2N)Tpi14 analogs are also more potent than Leu13 psi(CH2NH)Trp14 analogs in growth inhibition studies using Swiss 3T3 cells. The two best bombesin antagonists of this series, [D-Trp6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3415) and [Tpi6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3440), inhibited GRP-stimulated growth of Swiss 3T3 cells with IC50 values less than 1 nM. RC-3440 was also active in vivo, suppressing GRP(14-27)-stimulated serum gastrin secretion in rats. Bombesin/GRP antagonists, such as RC-3440, containing the new reduced bond (CH2N) reported herein are very potent.     

1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli [Cleary et al. (1989) Biochemistry 28, 1884-1891]. The spectrum of t-PA kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identifies side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Assignment of signals arising from the His13, His48a, and His64 side chains, which are unique to t-PA kringle 2, was assisted by the availability of a His64----Tyr mutant. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka approximately 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Acid/base titration of aromatic singlets in the presence of L-lysine yields pKa* approximately 6.25 and approximately 4.41 for His13 and His64, respectively, and shows that the His48a imidazole group does not protonate down to pH* approximately 4.3. Thus, the His48a and His64 side chains are in solvent-shielded locations. As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigations on C-H...pi interactions in RNA binding proteins

We have investigated the roles played by C-Hcdots, three dots, centeredpi interactions in RNA binding proteins. There was an average of 55 C-Hcdots, three dots, centeredpi interactions per protein and also there was an average of one significant C-Hcdots, three dots, centeredpi interaction for every nine residues in the 59 RNA binding proteins studied. Main-chain to side-chain C-Hcdots, three dots, centeredpi interactions is the predominant type of interactions in RNA binding proteins. The donor atom contribution to C-Hcdots, three dots, centeredpi interactions was mainly from Phe, Tyr, Trp, Pro, Gly, Lys, His and Ala residues. The acceptor atom contribution to main-chain to side-chain C-Hcdots, three dots, centeredpi and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions was mainly from Phe and Tyr residues. On the contrary, the acceptor atoms of Trp residues contributed to all the four types of C-Hcdots, three dots, centeredpi interactions. Also, Trp contributed both donor and acceptor atoms in main-chain to side-chain, main-chain to side-chain five-member aromatic ring and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions. The secondary structure preference analysis of C-Hcdots, three dots, centeredpi interacting residues showed that, Arg, Gln, Glu, His, Ile, Leu, Lys, Met, Phe and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Trp and Val preferred to be in strand conformation. Long-range C-Hcdots, three dots, centeredpi interactions are the predominant type of interactions in RNA binding proteins. More than 50% of C-Hcdots, three dots, centeredpi interacting residues had a higher conservation score. Significant percentage of C-Hcdots, three dots, centeredpi interacting residues had one or more stabilization centers. Seven percent of the theoretically predicted stabilizing residues were also involved in C-Hcdots, three dots, centeredpi interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Identification of coding polymorphisms in human circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS in a multi-ethnic screening panel.

STUDY OBJECTIVE: In this study, the exonic regions of the circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS were re-sequenced and coding changes identified in a panel of 95 individuals varying in ethnicity. STUDY PARTICIPANTS: DNA screening panel consisting of 95 DNA samples (17 American Caucasians, 17 African Americans, 8 Ashkenazi Jews, 8 Chinese, 8 Japanese, 5 Mexican Indians, 8 Mexicans, 8 Northern Europeans, 8 Puerto Ricans, and 8 South Americans) selected from the Coriell Institute Human Variation Panel. RESULTS: In addition to coding changes already identified in the database dbSNP, novel coding changes were identified, including PER1: Pro37Ser, Pro351Ser, Gln988Pro, Ala998Thr; PER2: Leu83Arg, Leu157Leu, Thre174Ile, Phe400Phe, Pro822Pro, Ala828Thr, Ala861Val, Phe876Leu, Val883Met, Val903Ile, Ala923Pro; PER3: Pro67Pro, Val90Ile, His638His, Ala820Ala, Leu929Leu; ARNTL: Arg166Gln, Ser459Phe; CLOCK: Ala34Ala, Ser208Cys, Phe233Phe, Ser632Thr, Ser816Ser; TIMELESS: Met870Val and CRY2: His35His. No coding polymorphisms were identified in CRY1. CONCLUSIONS: Considerable genetic variation occurs within the coding region of the genes regulating circadian rhythm. Many of the non-synonymous coding polymorphisms could affect protein structure/function with the potential to affect molecular regulation of the sleep/wake cycle. Many of the potential functional effects could be ethnic group specific.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Study of the spatial structure of a cyclic analog of bradykinin in solution by two-dimensional NMR spectroscopy

H NMR resonances of [cyclo (9----18) Lys1, Gly6]bradykinin (CBK) in (CD3)2SO and H2O solution have been assigned by combined analysis of two-dimensional COSY and NOESY spectra. The presence of two slowly interchangeable conformers of CBK in (CD3)2SO is established, the minor conformer not exceeding 15% in the population. The minor conformer is absent from the aqueous solution, chemical shifts of the CBK and bradykinin NH and C alpha H protons differ insignificantly. The major CBK conformer contains at least two X-Pro trans-peptide groups and three amide protons NH Phe5, NH Arg9 and N zeta H Lys1 protected from solvent. A system of cross-peaks from the NOESY spectra of CBK in (CD3)2SO has been analysed and the maximum distance between backbone protons and neighbouring amino acid residues evaluated. The experimental data agree well with the assumed type II beta-bend in the sequence Pro2-Pro3-Gly4-Phe5. Spatial structure models for the backbone fragment 6-9 of CBK containing two intramolecular hydrogen bonds that involve the NH Arg9 and N zeta H Lys1 protons and the carbonyl groups of Phe5 and Gly4 are proposed.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.