The physical state of two model mutants of alpha-hemolysin (alpha HL), alpha HL(1-289), a carboxyl-terminal deletion mutant (CDM), and alpha HL(1-331), a carboxyl-terminal extension mutant (CEM), were examined in deta...
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The physical state of two model mutants of alpha-hemolysin (alpha HL), alpha HL(1-289), a carboxyl-terminal deletion mutant (CDM), and alpha HL(1-331), a carboxyl-terminal extension mutant (CEM), were examined in detail to identify the role of the carboxyl terminus in the folding and function of native alpha HL, Denatured alpha HL can be refolded efficiently with nearly total recovery of its activity upon restoration of nondenaturing conditions. Various biophysical and biochemical studies on the three proteins have revealed the importance of an intact carboxyl terminus in the folding of alpha HL. The CDM exhibits a marked increase in susceptibility to proteases as compared with alpha HL, alpha HL and CEM exhibit similar fluorescence emission maxima, and that of the CDM is red-shifted by 9 nm, which indicates a greater solvent exposure of the tryptophan residues of the CDM, In addition, the CDM binds 8-anilino-1-naphthalene sulfonic acid (ANS) and increases its fluorescence intensity significantly unlike alpha HL and GEM, which show marginal binding. The circular dichroism studies point that the CDM possesses significant secondary structure, but its tertiary structure is greatly diminished as compared with alpha HL, These data show that the CDM has several of the features that characterize a molten globule state. Experiments with freshly translated mutants, using coupled in vitro transcription and translation, have further supported our observations that deletion at the carboxyl terminus leads to major structural perturbations in the water-soluble form of alpha HL. The studies demonstrate a critical role of the carboxyl terminus of alpha HL in attaining the native folded state.
Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable o...
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Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alpha gamma and alpha delta subunit interfaces (K-D similar to 100 pM) but binds with markedly reduced affinity to the alpha epsilon interface (K-D similar to 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gamma Pro-175 and gamma Glu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gamma Glu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gamma Glu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin, Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.
A new neurotoxin BmK M2, toxic to both mammals and insects, with the strongest toxicity in the BmK toxin series, has been purified from the Chinese scorpion Buthus martensii Karsch and crystallized with MPD at pH 7.5....
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A new neurotoxin BmK M2, toxic to both mammals and insects, with the strongest toxicity in the BmK toxin series, has been purified from the Chinese scorpion Buthus martensii Karsch and crystallized with MPD at pH 7.5. The crystals are orthorhombic, belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 36.64, b = 36.95, c = 37.23 Angstrom. The structure was solved by molecular replacement and refined to R = 0.186 for all reflections to a resolution of 1.76 Angstrom. The whole sequence (64 residues) of BmK M2 was determined by crystallographic analysis based on high-resolution data and the homologous model of BmK M8. The refined BmK M2 structure shows a non-proline cis peptide bond between Pro9 and His10 which enables the C-terminal segment to adopt a conformation different to that of the weak toxin BmK M8. Recently, a mutation analysis had suggested that both the tenth residue and the C-terminus play key roles in receptor binding. Therefore, these features may be related to the binding selectivity of the group III alpha-like toxins. The charge changes of residues 8, 10, 18, 28, 55 and 59 from neutral or negative to positive or neutral, which leads to a positive electrostatic potential surface, may be responsible for the high toxicity of BmK M2.
Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pe...
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Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region, Sequential deletions of C-terminal residues suggested an inevitable significance of Ile(73) and Ile(74) for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.
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