Biomedical subjects

G Bulaj

Publications and source records attributed to G Bulaj.

12 recordsLinked to original sources

PubMed2003

Detergent-assisted oxidative folding of delta-conotoxins.

Conotoxins comprise a diverse group of disulfide-rich peptides found in venoms of predatory Conus species. The native conformation of these peptides is marginally stable in comparison with alternative conformations, often resulting in low folding yields. The oxidative folding of hydrophobic delta-conotoxins was found to produce less than 1% of the native peptide [Bulaj, G. et al. (2001) Biochemistry 40, 13201]. In order to identify factors that might improve folding yields, we screened a number of additives including water-soluble polymers, detergents and osmolytes for their ability to increase steady-state accumulation of the native delta-conotoxin PVIA. The presence of a non-ionic detergent Tween and low temperature appeared to be the most effective factors in improving the oxidative folding. The detergent was also effective in promoting folding of other hydrophobic delta-conotoxins. Based on our findings, we discuss a possible mechanism for detergent-assisted folding and the general applicability of this mechanism to facilitating the proper folding of hydrophobic, cysteine-rich peptides.

Amino Acid Sequence↗

PubMed2002

Conorfamide, a Conus venom peptide belonging to the RFamide family of neuropeptides.

A novel Conus peptide, conorfamide-Sr1, has been characterized. The sequence of the natural peptide was determined using standard Edman sequencing methods and mass spectrometry, and confirmed by chemical synthesis. The peptide has 12 amino acids and no cysteine residues. The following sequence was obtained: GPMGWVPVFYRF-NH(2). No other peptide from a vermivorous Atlantic Conus species has previously been characterized. Conorfamide-Sr1 belongs to the RFamide neuropeptide family, and is the first RFamide peptide to be found in any venom. The presence of conorfamide-Sr1 as a major peptide in Conus spurius venom suggests that Conus lineages in the Atlantic may have evolved novel Conus venom peptide families.

Amino Acid Sequence↗

PubMed2001

Delta-conotoxin structure/function through a cladistic analysis.

Delta-conotoxins are Conus peptides that inhibit inactivation of voltage-gated sodium channels. The suggestion that delta-conotoxins might be an essential component of the venoms of fish-hunting cone snails which rapidly immobilize their prey [Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151] has not been tested. On the basis of cDNA cloning, all of the fish-hunting Conus analyzed yielded at least one delta-conotoxin sequence. In addition, one delta-conotoxin isolated from the venom of Conus striatus had an amino acid sequence identical to that predicted from cDNA cloning. This new peptide exhibited properties of delta-conotoxins: it targeted sodium channels and potentiated action potentials by slowing channel inactivation. Homologous sequences of delta-conotoxins from two groups (clades) of related fish-hunting Conus species share consensus features but differ significantly from the two known delta-conotoxins from mollusc-hunting Conus venoms. Three large hydrophobic amino acids were conserved; analogues of the previously described delta-conotoxin PVIA with alanine substituted for the conserved amino acids F9 and I12 lost substantial biological activity. In contrast, both the T8A and K13A delta-conotoxin PVIA analogues, where substitutions were at nonconserved loci, proved to be biologically active. Taken together, our results indicate that a cladistic approach can identify amino acids critical for the activity of conotoxins and provide extensive information as to which amino acid substitutions can be made without significant functional consequences.

Action Potentials↗

PubMed2001

Mutational analysis of hydrogen bonding residues in the BPTI folding pathway.

Nine BPTI variants with replacements that remove one or more hydrogen bonds from the native protein were constructed, and the folding pathways of these proteins were determined by isolating and identifying the disulfide-bonded intermediates that accumulated during unfolding and refolding. The forward and reverse rate constants for the individual steps in the folding pathways for each protein were measured, providing a detailed description of the energetic effects of the substitutions. The native forms of eight of the nine variants were measurably destabilized, by 1-7 kcal/mol (1 cal=4.184 J), with an average effect of 1.6 kcal/mol per hydrogen bond removed. The folding pathways for the variants were found to be similar to that previously described for the wild-type protein, with the kinetically preferred mechanism involving intramolecular rearrangements of intermediates with two disulfide bonds. Some of the substitutions, however, significantly destabilized the major intermediates and broadened the distribution of species with one or two disulfide bonds, thus identifying residues that play important roles in stabilizing the normal intermediates and defining specificity in the folding process. The kinetic data also suggest that one residue, Asn43, may play a distinctive role in defining the BPTI folding mechanism. Replacement of this residue with either Gly or Ala appeared to stabilize the major transition states for folding and unfolding. In the native protein, the side-chain of Asn43 participates directly in the hydrogen bonding pattern of the central beta-sheet, and the kinetic behavior of the Asn43 variants suggests that the major energy barriers in folding and unfolding may be due in part to the steric constraints imposed by this structural element, together with those imposed by the chemical transition states for thiol-disulfide exchange.

Amino Acid Substitution↗

PubMed2001

Phi-values for BPTI folding intermediates and implications for transition state analysis.

Amino acid replacements were used to probe the roles of 14 sites in two well-characterized intermediates in the folding pathway of bovine pancreatic trypsin inhibitor (BPTI). One of these intermediates contains one of the three disulfides found in the native protein (30--51). NMR studies have shown that approximately two-thirds of this polypeptide has a native-like conformation. The other intermediate contains two native disulfides (30--51 and 5--55) and has a fully folded conformation. The phi-values for a majority of residues were <1, indicating that the native protein was significantly more destabilized than either intermediate even when the altered residue was located in a well-ordered region of the intermediate. These observations suggest that folding intermediates and transition states may generally be more structured than indicated by phi-values alone.

Amino Acid Substitution↗

PubMed2000

Isolation and characterization of a novel conus peptide with apparent antinociceptive activity.

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.

Amino Acid Sequence↗

PubMed2000

Conotoxins - new vistas for peptide therapeutics.

There are approximately 500 species of predatory cone snails within the genus Conus. They comprise what is arguably the largest single genus of marine animals alive today. It has been estimated that the venom of each Conus species has between 50 and 200 components. These highly constrained sulfur rich components or conotoxins represent a unique arsenal of neuropharmacologically active peptides that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes. Remarkable divergence occurs when cone snails speciate. Consequently, the complement of venom peptides in any one Conus species is distinct from that of any other species. Hence many thousands of peptides that modulate ion channel function are present within Conus venoms. Evolutionary pressures have afforded a "pre-optimized," structurally sophisticated library that has been "fine tuned" over 50 million years. The statistics associated with sampling such libraries bear testimony to the validity and feasibility of this strategy. Although approximately 100 conotoxin sequences have been published in the scientific literature, representing a mere 0.2 % of the estimated library size, this sample has already afforded a peptide of proven clinical utility and several pre-clinical leads for CNS disorders. Conus libraries represent a rich pharmacopoeia and the potential to "therapeutically mine" such a resource appears limitless. The paucity of synthetic methodologies necessary to achieve the regioisomeric folding patterns present in these native peptides precludes access to synthetic conotoxin libraries, further validating the overall "mining" strategy. In this article, we will present a pragmatic overview of the molecular diversity as well as the neurobiological mechanisms that define each major class of conotoxin.

Amino Acid Motifs↗

PubMed1999

Early events in the disulfide-coupled folding of BPTI.

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.

Amino Acid Substitution↗

PubMed1998

Ionization-reactivity relationships for cysteine thiols in polypeptides.

Thiol-disulfide exchange reactions are required for many aspects of cellular metabolism including the folding of disulfide-bonded proteins, electron transfer, and numerous regulatory mechanisms. To identify factors influencing the rates of these reactions in polypeptides, the reactivities of Cys thiols in 16 model peptides were measured. For each of the peptides, which contained single Cys residues with thiol pKas ranging from 7.4 to 9.1, the rates of exchange with four disulfide-bonded compounds were measured. In reactions with two of the disulfide reagents, cystine and 2-hydroxyethyl disulfide, the peptide thiols displayed Bronsted correlations between reaction rate and pKa similar to those observed previously with model compounds (betanuc = 0.5 and 0.3, respectively). For two reagents with net charges, oxidized glutathione and cystamine, however, the apparent Bronsted coefficients were 0 and 0.8, respectively. These observations are in striking contrast with those obtained with model compounds, for which the Bronsted coefficients for the nucleophilic thiolates are largely independent of the disulfide-containing compound. The differences in the apparent Bronsted coefficients can be largely accounted for by electrostatic interactions between charged groups on the peptides and disulfide reagents and demonstrate that such interactions can play a dominant role in determining the rates of thiol-disulfide exchange in biological molecules. The results presented here provide an improved basis for predicting the rates of these reactions and suggest ways in which differences in the rates of competing reactions can be either minimized, to simplify the analysis of disulfide-coupled folding reactions, or enhanced, to favor formation of particular disulfides.

Amino Acid Sequence↗

PubMed1995

Ligand-induced changes in the conformational stability of bovine trypsinogen and their implications for the protein function.

Bovine trypsinogen was used as a model protein for studying changes in the conformational stability induced by pH or binding of the calcium ion. Spectrophotometrically monitored thermal unfolding of trypsinogen and beta-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to beta-trypsin, trypsinogen exhibits lower enthalpy of denaturation delta Hden, higher denaturational heat capacity change delta Cp,den, but very similar temperature of denaturation Tden. pH-dependence of the conformational stability of the ligand-free trypsinogen, measured also by GdnCl-induced unfolding, is bell shaped with the maximum free energy of unfolding delta Gden = 10.9 kcal/mole at pH 5.5 (4.5 pH units below its isoelectric point). At pH 8.3 the conformational stability of the zymogen drops to delta Gden = 3.2 kcal/mole, but increases by delta delta Gden = 6.1 kcal/mole in the presence of Ca2+. This significant stabilization of the zymogen by the calcium ion is also pH-dependent. To assess the effect of Ca2+ on the trypsinogen molecule, the spectrophotometric titrations and NOESY spectra were carried out. Based on the structural analysis, the long range effects between Ca2+-->Ile73-->Trp141 and the interdomain His40-Asp194 ion pair are proposed to be partially responsible for trypsinogen stabilization. Additionally, the steady-state parameters for hydrolysis of the oligopeptide amide substrate catalysed by free trypsinogen, its complexes with Ca2+ and the IleVal dipeptide and by beta-trypsin were measured. It appears that in the pH range 5.5 to 8.3 the stability and the catalytic activity/ligand binding properties are fully separated. Whereas the deprotonation of His57 accounts for the increase of kcat/km parameter, deprotonation of His40 is involved in the huge decrease of the conformational stability. Similarly, a large stabilization by the calcium ion is not accompanied by changes in enzymatic activity. Presented data are encouraging for an enzyme design directed toward improved stability.

Animals↗

PubMed1994

Denaturation of free and complexed bovine trypsinogen with the calcium ion, dipeptide Ile-Val and basic pancreatic trypsin inhibitor (Kunitz).

Thermal and chemical denaturation has been used to probe changes in the thermodynamic stability of trypsinogen upon complexation with calcium ion and with ligands, which induce the conformational transition of the zymogen to the trypsin-like form. Chemical and thermal unfolding curves of ligand-free trypsinogen at pH 5.8 are cooperative and yielded the following stability parameters: the free energy change of denaturation delta Gden = 44.8 kJ/mol, the denaturation temperature = 65.7 degrees C, the enthalpy change of denaturation delta Hden at the denaturation temperature Tden = 607 kJ/mol and the heat capacity change of denaturation delta Cp,den = 12.4 kJ.mol-1.K-1. Fast phases of both unfolding and refolding of trypsinogen proceed on a time scale of seconds and fit to a single exponential. At pH 5.8, the calcium ion increases the conformational stability delta Gden by 7.1 kJ/mol, Tden by 2.6 K and delta Hden by 80 kJ/mol, but does not induce any substantial structural change in the trypsinogen molecule, as revealed by 1H-NMR spectra. The trypsin-like form of trypsinogen, generated by complexation of the zymogen with the dipeptide Ile-Val and/or basic pancreatic trypsin inhibitor (Kunitz), is characterized by increase of delta Hden by 134 kJ/mol and Tden by 2.5 K, which may be attributed to the additional energy required to disrupt the rigidified activation domain in the complexed trypsinogen.

Animals↗

PubMed1994

Single peptide bond hydrolysis/resynthesis in squash inhibitors of serine proteinases. 2. Limited proteolysis of Curcurbita maxima trypsin inhibitor I by pepsin.

Porcine pepsin hydrolyzes the Leu7-Met8 (P2'-P3') peptide bond in Cucurbita maxima trypsin inhibitor I (CMTI I) in the pH range 2.0-4.8. The reaction proceeds to equilibrium between intact CMTI I and its cleaved form. The pH-independent value of the equilibrium constant (Khyd0 = 0.78) indicates that both forms of the inhibitor have similar Gibbs energies. The pH dependence of this constant shows that the peptide bond hydrolysis does not perturb ionization constants of any preexistent groups. The same equilibrium values can also be reached from the cleaved inhibitor side through pepsin-catalyzed resynthesis of the Leu7-Met8 peptide bond. Catalytic rate constants for the forward (hydrolysis) and reverse (resynthesis) reactions are similar. Both catalytic rate constants are strongly pH dependent, approaching the highest values at pH 2.0. Michaelis constant values for hydrolysis and resynthesis reactions depend much less on pH and are within values typical for oligopeptide substrates of pepsin. The influence of the binding loop rigidity on slow proteolysis by pepsin and other proteinases is discussed.

Amino Acid Sequence↗