Towards the design and computational characterization of a membrane protein.

The design of a transmembrane four-helix bundle is described. We start with an idealized four-helix bundle geometry, then use statistical information to build a plausible transmembrane bundle. Appropriate residues are chosen using database knowledge on the sequences of membrane helices and loops, then the packing of the bundle core is optimized, and favorable side chain rotamers from rotamer libraries are selected. Next, we use explicit physical knowledge from biomolecular simulation force fields and molecular dynamics simulations to test whether the designed structure is physically possible. These procedures test whether the designed protein will indeed be alpha-helical, well packed and stable over a time scale of several nanoseconds in a realistic lipid bilayer environment. We then test a modeling approach that does not include sophisticated database knowledge about proteins, but rather relies on applying our knowledge of the physics that governs protein motions. This independent validation of the design is based on simulated annealing and restrained molecular dynamics simulation in vacuo, comparable to procedures used to refine NMR and X-ray structures.

The structure of anhydrous and hydrated dimyristoylphosphatidyl glycerol: a pressure tuning infrared spectroscopic study.

The addition of water to anhydrous phospholipids has profound effects on the intermolecular interactions and packing order of phospholipid molecules. These changes, as well as alterations induced by hydration in the orientation of the acyl chains, can be qualitatively assessed using high pressure infrared spectroscopy. Application of this technique to dimyristoylphosphatidyl glycerol (DMPG) showed that hydration causes major changes in (i) the orientation of the carboxyl groups in relation to the glycerol backbone; (ii) the degree of orientational disorder of the acyl chains and (iii) the proximity of neighbouring DMPG molecules. In addition, the frequency of the carbonyl group is shown to be strongly affected not only by changes in the hydrogen bond network, but also by subtle environmental perturbations induced by the conformation of nearby structural units.

High pressure FTIR study of interaction of melittin with dimyristoylphosphatidyl glycerol bilayers.

Infrared spectra of hydrated dimyristoylphosphatidyl glycerol (DMPG) and of aqueous dispersions of melittin and DMPG at peptide:lipid molar ratios of 1:10 and 1:4 were recorded as a function of pressure from atmospheric to 22 kbar. Spectral features corresponding to vibrations of the amide linkages in melittin and to various functional groups in DMPG (carbonyl, methlylene, phosphate) were monitored in order to investigate the structure and dynamics of melittin:DMPG dispersions. Melittin was found to cause conformational and orientational disordering of the acyl chains in DMPG bilayers. The magnitude of these disorders was higher for higher concentration of melittin in DMPG. The higher concentration of melittin was also found to disrupt the DMPG bilayers through interactions with the lipid head groups. Such disruption may be related to some of the biological properties of melittin.

Folding and unfolding of the protoxin from Bacillus thuringiensis: evidence that the toxic moiety is present in an active conformation.

The action of trypsin or papain on the 130-kDa crystal protein (protoxin) from Bacillus thuringiensis subsp. kurstaki HD-73 yields a 67-kDa proteinase-resistant toxic fragment (toxin) which is derived from the N-terminal half of the molecule. Sensitivity to proteolysis and fluorescence emission spectroscopy showed that the toxin unfolded to a much greater extent in 6 M guanidinium chloride (GuHCl) than in 8 M urea. Protoxin also unfolded extensively in 6 M GuHCl, whereas in 8 M urea only the C-terminal half of the molecule had unfolded extensively. Both unfolded protoxin and unfolded toxin refolded to their native and biologically active conformations. The biphasic unfolding observed for protoxin suggests that the C-terminal half of the molecule unfolded rapidly, whereas the N-terminal toxic moiety unfolded at a much slower rate, similar to that of the free 67-kDa toxin. A 67-kDa fragment, derived from the N-terminal half of the molecule, could be generated from the protoxin in the presence of either urea or GuHCl by treatment with proteinases. Compared to toxin in denaturants, this fragment was found to be more sensitive to proteolysis. However, on removal of the denaturants the fragment had the same proteinase resistance and cytolytic activity as native toxin. The increased proteinase sensitivity of the fragment generated in the presence of denaturants appears to be due to a perturbation in the conformation of the N-terminal toxic moiety. This perturbation is attributed to the unfolding of the C-terminal region of the protoxin prior to its proteolysis to yield the 67-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus thuringiensis crystal protein: effect of chemical modification of the cysteine and lysine residues.

The 16 cysteine residues of reduced protoxin from Bacillus thuringiensis subsp. kurstaki HD-73 can be quantitatively reacted with: (a) iodoacetic acid, to give carboxymethyl protoxin; (b) iodoacetamide, giving carbaminomethyl protoxin and (c) N-(beta-iodoethyl)trifluoroacetamide to give aminoethyl protoxin. The carboxymethyl derivative was found to be significantly more soluble at neutral pH values where both the native protoxin and the carbaminomethyl derivative exhibit low solubilities. At the alkaline pH values (pH 9.5-10.5) normally used to solubilize the crystal protein, the native protein was slightly more soluble than either the carboxymethyl or the carbaminomethyl derivatives. The aminoethyl derivative had an extremely low solubility at all pH values. Succinic anhydride reacted with only 35% of the lysine residues in both the carboxymethyl and the carbaminomethyl protoxin derivatives. Nonetheless, these succinylated protoxins exhibited significantly increased solubilities at neutral pH values. All the derivatives were found to retain full insecticidal activity toward spruce budworm (Choristeneura fufimerana) larvae. It is concluded that all the cysteine residues and modified lysine residues are on the surface of the protein and that derivatization does not alter the conformation of the solubilized protoxin.

Simple assay and extraction of periplasmic penicillinase in Escherichia coli.

Benzylpenicillin was clearly separated from benzylpenicilloic acid by ascending chromatography on a diethylaminoethyl cellulose paper using 0.1 M ammonium acetate as a solvent. Using this chromatographic system, penicillinase was assayed by measuring the formation of [14C]benzylpenicilloic acid from [14C]benzylpenicillin. This assay remedies the lack of specificity of the commonly used iodometric assays. Periplasmic penicillinase was released from Escherichia coli by suspension in a mixture of 1% phenethyl alcohol and 5 mM ethylenediaminetetraacetate (pH 7.0). This simple extraction method not only facilitates the assay of penicillinase in an E. coli culture, but will also be useful for large-scale purification of periplasmic penicillinase.

The toxic moiety of the Bacillus thuringiensis protoxin undergoes a conformational change upon activation.

Proteolytic processing of the 133-kDa crystal protein (protoxin) from Bacillus thuringiensis subsp. kurstaki yields a 67-kDa insecticidal toxin. Differential scanning calorimetry was used to investigate whether the toxic moiety in the protoxin molecule has the same conformation as activated toxin. Compared to protoxin, toxin gives rise to a more complex endotherm which extends over a 10 degrees C broader temperature range and contains a component occurring at a substantially higher temperature than any unfolding transition in the protoxin endotherm. It is concluded that the toxic moiety undergoes a conformational change upon activation in which the thermal stability of at least one of its domains is significantly increased.

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73.

The secondary structure of the toxin from Bacillus thuringiensis subsp. kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33-40% alpha-helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32-40% beta-sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both alpha-helical and beta-sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.

Improved peptide detection with matrix-assisted laser desorption/ionization mass spectrometry by trimethylation of amino groups.

Trimethyl alpha-amino derivatives of peptides (penta to deca) with a permanent positive charge on their alpha-amino groups were prepared by in vacuo reaction with iodomethane and subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Compared to the unmodified peptide, the signal intensity of the trimethyl alpha-amino derivative in MALDI-MS is increased by at least an order of magnitude. Similarly, an octapeptide with a trimethylated epsilon-amino group derived from the solitary lysine residue of the B-chain of insulin also shows the same relative increase in signal intensity. Another advantage of the in vacuo methylation procedure is that trimethylation of a peptide amino group can be carried out readily with a combination of isotopes (13)CH(3)I and (12)CH(3)I or CD(3)I and CH(3)I, yielding a doublet signal either 3 or 9 units apart, respectively. The presence or absence of such a doublet signal can be used as a criterion to discriminate between peptide and non-peptide signals in the mass spectrum.

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications.

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.