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O Prakash

Publications and source records attributed to O Prakash.

At least 37 records · Page 2Linked to original sources
PubMed1999

Structure of a paralytic peptide from an insect, Manduca sexta.

Paralytic peptide 1 (PP1) from a moth, Manduca sexta, is a 23-residue peptide (Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu-Arg-Thr-Ala-Asp-Gly-Arg -Cys-Lys-Pro-Thr-Phe) that was first found to have paralytic activity when injected into M. sexta larvae. Recent studies demonstrated that PP1 also stimulated the spreading and aggregation of a blood cell type called plasmatocytes and inhibited bleeding from wounds. We determined the solution structure of PP1 by two-dimensional 1H NMR spectroscopy to begin to understand structural-functional relationships of this peptide. PP1 has an ordered structure, which is composed of a short antiparallel beta-sheet at residues Tyr11-Thr14 and Arg18-Pro21, three beta turns at residues Phe3-Gly6, Ala8-Tyr11 and Thr14-Gly17, and a half turn at the carboxyl-terminus (residues Lys20-Phe23). The well-defined secondary and tertiary structure was stabilized by hydrogen bonding and side-chain hydrophobic interactions. In comparison with two related insect peptides, whose structures have been solved recently, the amino-terminal region of PP1 is substantially more ordered. The short antiparallel beta-sheet of PP1 has a folding pattern similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF). Therefore, PP1 may interact with EGF receptor-like molecules to trigger its different biological activities.

Amino Acid Sequence↗
PubMed1999

The venous hum.

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Anemia↗
PubMed1998

Rigid phencyclidine analogues. Binding to the phencyclidine and sigma 1 receptors.

Three phencyclidine (PCP) analogues possessing a highly rigid carbocyclic structure and an attached piperidine ring which is free to rotate were synthesized. Each analogue has a specific fixed orientation of the ammonium center of the piperidinium ring to the centrum of the phenyl ring. The binding affinities of the rigid analogues 1-piperidino-7,8-benzobicyclo[4.2.0]octene (14), 1-piperidinobenzobicyclo[2.2.1]heptene (16), and 1-piperidinobenzobicyclo[2.2.2]octene (13) for the PCP receptor ([3H]TCP) and th-receptor (NANM) were determined. The three analogues show low to no affinity for the PCP receptor but good affinity for the th-receptor and can be considered th-receptor selective ligands with PCP/th ratios of 13, 293, and 368, respectively. The binding affinities for the th-receptor are rationalized in terms of a model for the th-pharmacophore.

Animals↗
PubMed1998

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.

Amino Acid Sequence↗
PubMed1998

NMR analysis of a potent decapeptide agonist of human C5a anaphylatoxin.

A NMR investigation in H2O, TFE and DMSO of a conformationally constrained, potent decapeptide agonist of human C5a, YSFKDMPLaR (C5a65-74, Y65, F67, P71, D-Ala73) showed that its N-terminal region (YSFKD) exhibited an extended backbone conformation in H2O and a more twisted conformation in both TFE/H2O (30:70, v/v; referred to as TFE) and DMSO. The C-terminal region (MPLaR) of the peptide adopted compact, turn-like structures. In H2O, the C-terminal region adopted a type II beta-turn or a distorted type V/II beta-turn involving residues PLaR. In the distorted type V/II beta-turn, Leu72 exhibited a conformation typical of a type V beta-turn, whereas D-Ala73 exhibited a conformation typical of a type II beta-turn. The distorted type V/II beta-turn overlapped with an inverse gamma-turn involving residues MPL. In DMSO, the C-terminal region had the analogous inverse gamma-turn and the V/II beta-turn found in H2O. In many of the DMSO structures, two inverse gamma-turns in the MPL and PLa positions formed a double-inverse gamma-turn. None of the turns observed in H2O were present in TFE. However, in TFE, the PLa residues formed an inverse gamma-turn. Overall, the turn-like structural motifs in the C-terminal region of the peptide in both H2O and DMSO (but not in TFE) agreed with the biologically important conformations obtained earlier by the structure-function analysis of a panel of C5a agonist peptides. These motifs may represent key structural elements important for C5a agonist activity and may be used to design the next generation of C5a agonist and antagonist analogues.

Amino Acid Sequence↗
PubMed1998

A study of inhibition of urease from seeds of the water melon (Citrullus vulgaris).

Urease from seeds of the water melon was found to be inhibited by various salts of sodium. Sodium fluoride strongly inhibited the activity in the low urea concentration range. The enzyme was also inhibited by a high concentration of urea which was completely abolished in the presence of 10 mM sodium fluoride. Time-dependent inactivation of urease with iodoacetic acid, N-ethylmaleimide and p-hydroxymercuribenzoate exhibited biphasic kinetics in which half of the initial activity was lost in the fast phase and the remainder in a slow phase. Each phase exhibited first-order kinetics. These observations are suggestive of the existence of half-and-half distribution of sites.

Enzyme Activation↗
PubMed1998

The human immunodeficiency virus type I Tat protein potentiates ethanol-induced neutrophil functional impairment in transgenic mice.

Neutropenia and impairment of neutrophil function are commonly observed in patients with human immunodeficiency virus (HIV) infections. The HIV regulatory protein Tat is known to exert immunosuppressive effects. Alcohol is known also to be an immunosuppressive factor, and as alcohol abuse is common among HIV infected hosts, both factors may interact in an additive or synergistic fashion to further impair the host defenses of these patients. In order to test this possibility, endotoxin-induced neutrophil beta2-integrins CD11b and CD18 expression, phagocytosis, and hydrogen peroxide generation were examined in normal and HIV-1 Tat-transgenic mice in the absence and presence of ethanol intoxication. Acute ethanol intoxication was induced in mice (body weight of 25+/-1 g) by an intraperitoneal (ip) injection of ethanol (3 g/kg, 20% in normal saline). Thirty min later, the animals were given an ip injection of endotoxin (20 microg in 0.2 ml of saline/mouse). Vehicle-treated controls received an ip injection of saline without ethanol or endotoxin. Two hr after endotoxin administration, the animals were killed to determine neutrophil functions with flow cytometry. The baseline expression of CD11b and CD18 was similar in normal nontransgenic and Tat-transgenic mice. Endotoxin administration significantly up-regulated CD11b and CD18 expression in normal mice. This up-regulation was suppressed in Tat-transgenic mice. Ethanol intoxication inhibited endotoxin-induced CD11b and CD18 expression in normal mice and totally abolished endotoxin-induced CD11b and CD18 expression in Tat-transgenic mice. Neutrophil phagocytic activity in normal and Tat-transgenic mice was similar. Ethanol intoxication produced a similar inhibition of phagocytosis in both study groups. Endotoxin suppressed phagocytosis in normal mice, and this suppression was more pronounced in Tat-transgenic mice. Spontaneous and phorbol myristate acetate (PMA)-stimulated hydrogen peroxide generation by circulating neutrophils (PMNs) were similar in normal and Tat-transgenic mice. Neither ethanol nor endotoxin affected hydrogen peroxide generation by PMNs. These data show that both Tat and alcohol significantly impair PMN function, and this may be a mechanism leading to the increased susceptibility of the HIV-infected host to bacterial infection.

AIDS-Related Opportunistic Infections↗
PubMed1997

The human immunodeficiency virus type 1 Tat protein potentiates zidovudine-induced cellular toxicity in transgenic mice.

3'-Azido-2',3'-dideoxythymidine (AZT, zidovudine) is the principal antiretroviral agent in the treatment of AIDS. Although beneficial, AZT remains restricted for human usage because of its severe toxic effects. We examined the AZT sensitivity in transgenic mice expressing HIV-1 one-exon-encoded 72 amino acid Tat (Tat72) and full-length 86 amino acid Tat (Tat86) proteins. Administration of AZT (1 mg/ml) in drinking water for 1 week resulted in a three- to fourfold decrease in hematopoietic progenitors from bone marrow in Tat mice compared to AZT-treated nontransgenic controls as determined by erythroid and granulocyte/macrophage colony-forming unit assays. In liver and thymus, two of the tissues examined, AZT treatment of Tat mice resulted in as much as 80-90% suppression of Mn-superoxide dismutase (Mn-SOD) activity. Other parameters associated with loss of Mn-SOD such as increase in carbonyl proteins and decrease of sulfhydryl content were also significantly enhanced by AZT in Tat mice. Our in vivo study suggests that AZT therapy is associated with oxidative damage affecting cellular functions in several tissues and that Tat is one of the contributory factors in AZT-induced toxicities. The findings of AZT-induced oxidative damage may help to improve the therapeutic index of AZT and other related drugs in AIDS patients.

Animals↗
PubMed1997

Structure-based design of a general class of mechanism-based inhibitors of the serine proteinases employing a novel amino acid-derived heterocyclic scaffold.

We describe in this paper the structure-based design of a general class of heterocyclic mechanism-based inhibitors of the serine proteinases that embody in their structure a novel peptidomimetic scaffold (1,2,5-thiadiazolidin-3-one 1,1-dioxide). Sulfone derivatives of this class (I) were found to be time-dependent, potent, and highly efficient irreversible inhibitors of human leukocyte elastase, cathepsin G, and proteinase 3. The partition ratios for a select number of inhibitors were found to range between 0 and 1. We furthermore demonstrate that these inhibitors exhibit remarkable enzyme selectivity that is dictated by the nature of the P1 residue and is consistent with the known substrate specificity reported for these enzymes. Thus, inhibitors with small hydrophobic side chains were found to be effective inhibitors of elastase, those with aromatic side chains of cathepsin G, and those with a basic side chain of bovine trypsin. Taken together, the findings cited herein reveal the emergence of a general class of stable mechanism-based inhibitors of the serine proteinases which can be readily synthesized using amino acid precursors. Biochemical and high-field NMR studies show that the interaction of this class of inhibitors with a serine proteinase results in the formation of a stable acyl complex(es) and the release of benzenesulfinate, formaldehyde, and a low molecular weight heterocycle. The data are consistent with initial formation of a Michaelis-Menten complex, acylation of Ser195, and tandem loss of the leaving group. The initial HLE-inhibitor complex reacts with water generating formaldehyde and a stable HLE-inhibitor complex. Whether the initial HLE-inhibitor complex also reacts with His57 to form a third complex is not known at this point. The desirable salient parameters associated with this class of inhibitors, including the expeditious generation of structurally diverse libraries of inhibitors based on I, suggest that this class of mechanism-based inhibitors is of general applicability and can be used in the development of inhibitors of human and viral serine proteinases of clinical relevance.

Amino Acids↗
PubMed1997

Interleukin-4 in the treatment of AIDS-related Kaposi's sarcoma.

PURPOSE: To define the safety and toxicity of interleukin-4 (IL-4) when administered subcutaneously in patients with AIDS-related Kaposi's sarcoma (AIDS-KS); to evaluate the effect of IL-4 on immunologic and virologic parameters; and to preliminarily assess the response rate of IL-4 in AIDS-KS. PATIENTS AND METHODS: Eighteen patients with mucocutaneous, non-visceral AIDS-KS were treated with IL-4 at a dose of 1 mcg/kg subcutaneously, daily until unacceptable toxicity or for a maximum period of six months. Twelve (66%) patients had extensive mucocutaneous disease with over 25 lesions. Ten patients had received prior systemic chemotherapy. Seventeen had CD4+ lymphocyte counts less than 200/mm3. RESULTS: The most common adverse effects included headache in 78%, fever in 56%, chills in 44%, and edema in 44%. Hematologic toxicities consisted of grade 4 neutropenia (less than 500/mm3) in 33%, mild anemia in 22%. Transient elevation of liver enzymes was noted in 17%. A transient elevation in CD4+ lymphocyte counts occurred during the first two weeks of therapy. Four of eleven patients tested showed marked decline in plasma HIV RNA after four weeks. Partial remission was observed in one patient, lasting six months. Three other patients (17%) had stable disease: 7 weeks in one patient, and 10 weeks in each of the two other patients. CONCLUSION: Grade 4 neutropenia (absolute neutrophil count < 500/mm3) was the most common hematologic adverse effect with IL-4 in patients with AIDS-KS. In contrast to in vitro findings, there was a decrease in plasma HIV RNA after four weeks of IL-4 therapy in the majority of patients tested. IL-4 produced minimal anti-tumor effects in AIDS-KS with one partial remission in a patient with CD4 lymphocyte counts over 200/mm3. Further studies of IL-4 in AIDS-KS may be considered in patients with better immune status.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini.

The solution structure and internal dynamics of the reactive-site (Lys44-Asp45 peptide bond) hydrolyzed form of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V*) were characterized by the application of two-dimensional 1H-15N NMR methods to the uniformly 15N-labeled protein. The 1H-15N chemical shift correlation spectra of rCMTI-V* were assigned, and the chemical shift data were compared with those available for rCMTI-V [Liu, J., Prakash, O., Cai, M., Gong, Y., Huang, Y., Wen, L., Wen, J. J., Huang, J.-K., & Krishnamoorthi, R. (1996) Biochemistry 35, 1516-1524] and CMTI-V* [Cai, M., Gong, Y., Prakash, O., & Krishnamoorthi, R. (1995) Biochemistry 34, 12087-12094] for which three-dimensional solution structures have been determined. It was deduced that the solution structure of rCMTI-V* was almost the same as that of CMTI-V*. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and ¿1H¿-15N steady-state heteronuclear Overhauser effects were measured for the peptide NH units and arginine and tryptophan N epsilon H groups in rCMTI-V*, and the model-free parameters [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570] were computed. Most of the backbone of rCMTI-V* is found to be highly constrained (S2 = 0.85), including the N-terminal residues 3-6 (S2 = 0.77). Residues 39-44, forming the C-terminal fragment of the binding loop, exhibit increased mobility (S2 = 0.51); however, the N-terminal segment (residues 46-48) retains rigidity as in the intact form (S2 = 0.83). The S2 values, 0.78 and 0.59, respectively, of Arg50 and Arg52 side chain NHs provide evidence not only for the conservation of the Arg hydrogen-bonds with the binding loop segments but also for the difference in strength between them. This is consistent with the earlier observation made from a study of rCMTI-V at two different pHs and its R50 and R52 mutants [Cai, M., Huang, Y., Prakash, O., Wen, L., Dunkelbarger, S. P., Huang, J.-K., Liu, J., & Krishnamoorthi, R. (1996) Biochemistry 35, 4784-4794]. The dynamical results suggest the mainchain oxygen atom of Asp45 as the hydrogen bond acceptor of Arg50. Residues Trp9 and Trp54, which interact with many others in the protein scaffold and the binding loop region, respectively, remain rigid in the cleaved inhibitor with the S2 values of 0.84 and 0.71 determined for their respective N epsilon Hs. The internal dynamics of rCMTI-V* was compared with that of the noncovalent complex formed between the two fragments of reactive-site-hydrolyzed chymotrypsin inhibitor-2 from barley seeds [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233], another potato I family inhibitor that lacks the Cys3-Cys48 disulfide present in rCMTI-V*.

Arginine↗
PubMed1996

Aberrant regulation of cytokines in HIV-1 TAT72-transgenic mice.

The trans-activator of transcription or TAT gene from HIV-1 encodes a protein that increases the processivity of transcription from the HIV-1 genome. TAT protein can also affect cellular processes in the absence of its ribonucleic HIV target sequence trans activation response element and may be responsible for some aspects of HIV pathogenesis apart from infectious virus or other viral gene products. We have previously shown that TAT72 decreases CTL activity in TAT72-transgenic mice, and we now demonstrate aberrant regulation of mitogen-elicited IL-2 at both transcriptional and translational levels. In contrast, alloantigen stimulation resulted in increased IL-6 and IL-10 production in the TAT72-transgenic mice. Con A-stimulated cultures of splenic lymphocytes from TAT72-transgenic mice do not undergo clonal proliferation of CD4+ cells as compared with CD8+ cells monitored over 72 h. These results suggest that TAT is sufficient to induce some pathology associated with AIDS and is a potent immunologic manipulator apart from its function as trans-activator.

Animals↗
PubMed1996

Characterization of products from the reactions of N-acetyldopamine quinone with N-acetylhistidine.

When insects harden or sclerotize their exoskeletons, quinones of N-acetylated catecholamines such as N-acetyldopamine (NADA) undergo nucleophilic addition reactions with amino acids such as histidine in cuticular proteins. To determine the products that might form when this type of reaction occurs during cuticle sclerotization, the reactions between electrochemically prepared NADA quinone and N-acetylistidine (NAcH), a protein model nucleophile, have been investigated at pH 7. Two major products, 6-[N-(N-acetylhistidyl)]-N-acetyldopamine and 2-[N-(N-acetylhistidyl)]-N-acetyldopamine, were purified by semipreparative reversed-phase liquid chromatography and identified by mass spectrometry and nuclear magnetic resonance spectroscopy. The relative molar ratio of the C(6) mono-addition adduct to the C(2) mono-addition adduct is 87:13. UV/vis spectroscopic analysis shows that both products have an absorption maximum at 284 nm. Cyclic voltammetry shows that these adducts are oxidized less readily than NADA.

Benzoquinones↗
PubMed1996

Differential modulation of binding loop flexibility and stability by Arg50 and Arg52 in Cucurbita maxima trypsin inhibitor-V deduced by trypsin-catalyzed hydrolysis and NMR spectroscopy.

The side chains of Arg50 and Arg52 iin Cucurbita maxima trypsin inhibitor-V (CMTI-V) anchor the binding loop to the scaffold region [Cai, M., Gong, Y., Kao, J.L-F., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The consequences of these hydrogen-bonding and electrostatic interactions on the conformational flexibility and stability of the binding loop were evaluated by trypsin-catalyzed hydrolysis of CMTI-V mutants, in which each of the arginines was individually replaced with Ala, Lys, or Gln by genetic engineering methods. All mutants exhibited significantly increased vulnerability to the protease attack at many sites, including the reactive-site (Lys44-Asp45 peptide bond), with the R50 mutants showing much more pronounced effects than the R52 counterparts. For CmTI-V and the mutants studied, a qualitative correlation was inferred between binding loop flexibility and retention time on a reverse-phase high-pressure liquid chromatography C-18 column. The R50 mutants were found to be more flexible than the corresponding R52 versions. These results demonstrate that Arg50 contributes more to the stability and function of CMTI-V. The differing strengths of the hydrogen bonds made by Arg50 and Arg52 were characterized by determining the internal dynamics of their side chains at pH 5.0 and 2.5: 15N NMR longitudinal and transverse relaxation rates and 15N-1H nuclear Overhauser effect (NOE) enhancements were measured for the main-chain and side-chain NH groups in 15N-labeled recombinant CMTI-V (rCMTI-V) and the model-free parameters [Lipari, G., & Szabo, A.(1982) J. Am. Chem. Soc. 104, 4546-59; 4559-4570] were calculated. At both pH 5.0 and 2.5, the arginines at positions 26, 47, 58 and 66 are found to be highly mobile, as the caluculated general order parameters, S2 values, of their NepsilonH groups fall in the range 0.03-0.18. The corresponding values for Arg50 amd Arg52 are 0.73 and 0.63, respectively, at pH 5.0, thus confirming that the two arginines are rigid and hydrogen bonded. At pH 2.5, these hydrogen bonds are still retained with Arg50 appearing to be more restrained (S2 = 0.71) than Arg52 (S2 = 0.56). This is consistent a greater contribution by Arg50 to the conformational stability of the reactive-site loop in CMTI-V. The results also indicate that the Arg50 and Arg52 side chains are not hydrogen-bonded to carboxylate groups, which would be protonated at pH 2.5 and, hence, unavailable for hydrogen-bonding interactions. The overall folding of rCMTI-V appears not to be significantly affected by the pH change, as indicated by comparisons of 1H and 15N chemical shifts, sequential NOE cross-peaks, and S2 values of the backbone atoms, and the conserved side-chain dynamics of Trp9 and Trp54--residues that are involved in hydrophobic and hydrogen-bonding interactions with others in the protein core and the binding loop, respectively.

Arginine↗
PubMed1996

Endogenous mouse mammary tumor virus Mtv-17 is involved in Mtv-2-induced tumorigenesis in GR mice.

Mtv-2 is an endogenous mouse mammary tumor virus (MMTV) that is responsible for the induction of mammary gland tumors in the high mammary gland tumor-incidence strain GR. GR animals inherit four different endogenous MMTVs in addition to Mtv-2: Mtv-3, Mtv-7, Mtv-8 and Mtv-17. In this study we analyzed the involvement of these nonpathogenic endogenous proviruses in the mammary gland tumors caused by Mtv-2. We showed that Mtv-17 is expressed in the mammary gland of GR mice, efficiently packaged into virions, and shed into milk. DNA isolated from both mammary gland tumors and the nonmalignant mammary gland tissues of GR mice contained amplified copies of both newly acquired Mtv-2 and recombinant proviruses with the env gene derived from Mtv-17. A small percentage of these tumors contained predominantly recombinant viruses. These findings suggest a role for Mtv-17 in the mammary gland tumors induced by Mtv-2 in GR mice.

Animals↗
PubMed1996

Solution structure and backbone dynamics of recombinant Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy.

The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.

Magnetic Resonance Spectroscopy↗
PubMed1996

Design, synthesis, and in vitro inhibitory activity toward human leukocyte elastase, cathepsin G, and proteinase 3 of saccharin-derived sulfones and congeners.

The inhibitory activity toward human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR 3) of a series of saccharin derivatives having a sulfinate leaving group was investigated. The results of this study revealed that (a) inhibitory activity is dependent on the nature and pKa of the leaving group, and (b) the synthesized saccharin derivatives exhibit selective inhibition toward HLE and PR 3, with low or no activity toward cathepsin G. The results of exploratory biochemical, HPLC and high-field 13C NMR studies are also described.

Autoantigens↗