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1.
FEBS J ; 280(11): 2675-87, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23552119

ABSTRACT

This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.


Subject(s)
Calmodulin/chemistry , Binding Sites , Calcium/chemistry , Calcium/metabolism , Calcium Signaling , Calmodulin/metabolism , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Humans , Maleimides/chemistry , Models, Molecular , Osmolar Concentration , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Conformation , Protein Denaturation , Protein Structure, Tertiary , Quinolinium Compounds/chemistry , Static Electricity
2.
J Nat Prod ; 73(8): 1411-21, 2010 Aug 27.
Article in English | MEDLINE | ID: mdl-20687534

ABSTRACT

Two related peptide metabolites, one a cyclic depsipeptide, hoiamide B (2), and the other a linear lipopeptide, hoiamide C (3), were isolated from two different collections of marine cyanobacteria obtained in Papua New Guinea. Their structures were elucidated by combining various techniques in spectroscopy, chromatography, and synthetic chemistry. Both metabolites belong to the unique hoiamide structural class, characterized by possessing an acetate extended and S-adenosyl methionine modified isoleucine unit, a central triheterocyclic system comprised of two alpha-methylated thiazolines and one thiazole, and a highly oxygenated and methylated C-15 polyketide unit. In neocortical neurons, the cyclic depsipeptide 2 stimulated sodium influx and suppressed spontaneous Ca(2+) oscillations with EC(50) values of 3.9 microM and 79.8 nM, respectively, while 3 had no significant effects in these assays.


Subject(s)
Cyanobacteria/chemistry , Depsipeptides/isolation & purification , Lipopeptides/isolation & purification , Neurotoxins/isolation & purification , Animals , Depsipeptides/chemistry , Depsipeptides/pharmacology , Female , Humans , Lipopeptides/chemistry , Lipopeptides/pharmacology , Marine Biology , Mice , Molecular Structure , Neurons/drug effects , Neurotoxins/chemistry , Neurotoxins/pharmacology , Nuclear Magnetic Resonance, Biomolecular , Papua New Guinea , Pregnancy
3.
J Nat Prod ; 71(1): 22-7, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18163584

ABSTRACT

Investigation of a Symploca sp. from Papua New Guinea has led to the isolation of symplocamide A (1), a potent cancer cell cytotoxin, which also inhibits serine proteases with a 200-fold greater inhibition of chymotrypsin over trypsin. The complete stereostructure of symplocamide A was determined by detailed NMR and MS analysis as well as chiral HPLC analysis of the component amino acid residues. The presence of several unusual structural features in symplocamide A provides new insights into the pharmacophore model for protease selectivity in this drug class and may underlie the potent cytotoxicity of this compound to H-460 lung cancer cells (IC50=40 nM) as well as neuro-2a neuroblastoma cells (IC50=29 nM).


Subject(s)
Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Chymotrypsin/antagonists & inhibitors , Cyanobacteria/chemistry , Cytotoxins/isolation & purification , Cytotoxins/pharmacology , Depsipeptides/isolation & purification , Depsipeptides/pharmacology , Marine Toxins/isolation & purification , Marine Toxins/pharmacology , Animals , Antineoplastic Agents/chemistry , Cytotoxins/chemistry , Depsipeptides/chemistry , Drug Screening Assays, Antitumor , Humans , Leishmania donovani/drug effects , Marine Toxins/chemistry , Molecular Structure , Papua New Guinea , Plasmodium falciparum/drug effects , Trypanosoma cruzi/drug effects
4.
J Mol Biol ; 358(3): 870-81, 2006 May 05.
Article in English | MEDLINE | ID: mdl-16530223

ABSTRACT

Reconstitution studies of a protein from domain fragments can furnish important insights into the distinctive role of particular domain interactions and how they affect biophysical properties important for function. Using isothermal titration calorimetry (ITC) and a number of spectroscopic and chromatographic tools, including CD, fluorescence and NMR spectroscopy, size-exclusion chromatography and non-denaturing agarose gel electrophoresis, we have investigated the reconstitution of the ubiquitous Ca2+-sensor protein calmodulin (CaM) and its globular domains from fragments comprising one or two EF-hands. The studies were carried out with and without the target peptide from smooth muscle myosin light chain kinase (smMLCKp). The CaM-target complex can be reconstituted from the three components consisting of the target peptide and the globular domains TR1C and TR2C. In the absence of peptide, there is no evidence for association of the globular domains. The globular domains can further be reconstituted from their corresponding native subdomains. The dissociation constant, K(D), in 2 mM Tris-HCl (pH 7.5), for the subdomain complexes, EF1:EF2 and EF3:EF4, was determined with ITC to 9.3 x 10(-7) M and 5.9 x 10(-8) M, respectively. Thus, the affinity between the two C-terminal subdomains, located within TR2C, is stronger by a factor of 16 than that between the corresponding subdomains within TR1C. These observations are corroborated by the spectroscopic and chromatographic investigations.


Subject(s)
Calmodulin/chemistry , Calmodulin/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Amino Acid Sequence , Animals , Calmodulin/genetics , Calorimetry , Chickens , Chromatography, Gel , Circular Dichroism , Conserved Sequence , EF Hand Motifs , Humans , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Myosin-Light-Chain Kinase/metabolism , Peptide Fragments/genetics , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Temperature , Titrimetry
5.
J Med Chem ; 47(24): 5953-61, 2004 Nov 18.
Article in English | MEDLINE | ID: mdl-15537350

ABSTRACT

Despite the availability of large amounts of data for HIV-protease inhibitors and their effectiveness with wild type and resistant enzyme, there is limited knowledge about how this and other information can be systematically applied to the development of new antiviral compounds. To identify in vitro parameters that correlate with the efficacy of HIV inhibitors in cell culture, the relationships between inhibition, interaction kinetic, and cell culture parameters for HIV-1 protease inhibitors were analyzed. Correlation, cluster, and principal component analysis of data for 37 cyclic and linear compounds revealed that the affinities (K(D)) determined from SPR-biosensor binding studies correlated better to cell culture efficacy (ED(50)) than that of the inhibition constants (K(i)), indicating that the conventional use of K(i) values for structure-activity relationship analysis of HIV-1 inhibitors should be seriously reconsidered. The association and dissociation kinetic rate constants (k(on) and k(off)) alone showed weak correlations with ED(50) values. However, ED(50) values were most related to the free enzyme concentration in the viral particle ([E]), calculated from the rate constants and the total enzyme concentration in a viral particle. A structure-activity relationship analysis of the current data set was found to be valid for all classes of compounds analyzed. In summary, use of affinity, based on interaction kinetic rate constants, rather than inhibition constants, and theoretical consideration of the physiological conditions in the virus particle provide improved structure-activity relationship analysis of HIV-1 protease inhibitors.


Subject(s)
HIV Protease Inhibitors/chemistry , HIV Protease/chemistry , HIV-1/chemistry , Biosensing Techniques , Cells, Cultured , Cluster Analysis , HIV Protease/metabolism , HIV Protease Inhibitors/classification , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Kinetics , Principal Component Analysis , Quantitative Structure-Activity Relationship , Virus Replication/drug effects
6.
J Mol Recognit ; 17(2): 106-19, 2004.
Article in English | MEDLINE | ID: mdl-15027031

ABSTRACT

Interaction kinetic and thermodynamic analyses provide information beyond that obtained in general inhibition studies, and may contribute to the design of improved inhibitors and increased understanding of molecular interactions. Thus, a biosensor-based method was used to characterize the interactions between HIV-1 protease and seven inhibitors, revealing distinguishing kinetic and thermodynamic characteristics for the inhibitors. Lopinavir had fast association and the highest affinity of the tested compounds, and the interaction kinetics were less temperature-dependent as compared with the other inhibitors. Amprenavir, indinavir and ritonavir showed non-linear temperature dependencies of the kinetics. The free energy, enthalpy and entropy (DeltaG, DeltaH, DeltaS) were determined, and the energetics of complex association (DeltaG(on), DeltaH(on), DeltaS(on)) and dissociation (DeltaG(off), DeltaH(off), DeltaS(off)) were resolved. In general, the energetics for the studied inhibitors was in the same range, with the negative free energy change (DeltaG < 0) due primarily to increased entropy (DeltaS > 0). Thus, the driving force of the interaction was increased degrees of freedom in the system (entropy) rather than the formation of bonds between the enzyme and inhibitor (enthalpy). Although the DeltaG(on) and DeltaG(off) were in the same range for all inhibitors, the enthalpy and entropy terms contributed differently to association and dissociation, distinguishing these phases energetically. Dissociation was accompanied by positive enthalpy (DeltaH(off) > 0) and negative entropy (DeltaS(off) < 0) changes, whereas association for all inhibitors except lopinavir had positive entropy changes (DeltaS(on) > 0), demonstrating unique energetic characteristics for lopinavir. This study indicates that this type of data will be useful for the characterization of target-ligand interactions and the development of new inhibitors of HIV-1 protease.


Subject(s)
HIV Protease Inhibitors/chemistry , HIV-1 , Carbamates , Furans , Indinavir/chemistry , Kinetics , Lopinavir , Pyrimidinones/chemistry , Ritonavir/chemistry , Sulfonamides/chemistry , Thermodynamics
7.
Antiviral Res ; 58(3): 235-42, 2003 May.
Article in English | MEDLINE | ID: mdl-12767471

ABSTRACT

The kinetics of the interaction between drug-resistant variants of HIV-1 protease (G48V, V82A, L90M, I84V/L90M, and G48V/V82A/I84V/L90M) and clinically used inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) were determined using biosensor technology. The enzyme variants were immobilized on a biosensor chip and the association and dissociation rate constants (k(on) and k(off)) and affinities (K(D)) for interactions with inhibitors were determined. A unique interaction kinetic profile was observed for each variant/inhibitor combination. Substitution of single amino acids in the protease primarily resulted in reduced affinity through increased k(off) for the inhibitors. For inhibitors characterized by fast association rates to wild-type protease (ritonavir, amprenavir, and indinavir), additional substitutions resulted in a further reduction of affinity by a combination of decreased k(on) and increased k(off). For inhibitors characterized by slow dissociation rates to wild-type enzyme (saquinavir and nelfinavir), the decrease of affinity conferred by additional mutations was attributed to increased k(off) values. Development of resistance thus appears to be associated with a change of the distinctive kinetic parameter contributing to high affinity. Further inhibitor design should focus on improving the "weak point" of the lead compound, that being either k(on) or k(off).


Subject(s)
Drug Resistance, Viral , Genetic Variation , HIV Protease Inhibitors/metabolism , HIV Protease/metabolism , Amino Acid Substitution , Biosensing Techniques , HIV Protease/drug effects , HIV Protease/genetics , Humans , Kinetics , Models, Molecular , Surface Plasmon Resonance
8.
Protein Expr Purif ; 25(3): 363-71, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12182815

ABSTRACT

Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni(2+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K(M)) for catalysis and the inhibitory potencies (IC(50) and K(i)) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.


Subject(s)
Hepacivirus/enzymology , RNA Helicases/genetics , RNA Helicases/isolation & purification , Viral Nonstructural Proteins/isolation & purification , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetic Vectors , Glycerol/pharmacology , Hepacivirus/genetics , Inhibitory Concentration 50 , Molecular Sequence Data , RNA Helicases/biosynthesis , RNA, Viral/genetics , RNA, Viral/isolation & purification , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Nucleic Acid , Sodium Chloride/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
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