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The contributions of protein motions to the dielectric response in solvent-inaccessible regions of calmodulin are examined. Two-pulse and three-pulse stimulated-emission experiments are used to examine the Stokes shift dynamics of coumarins in their ground and excited states. The results allow an evaluation of linear response theory. For coumarin 153, the ground-and excited-state solvation dynamics are similar to those for acetonitrile and methanol solvents. The solvation of coumarin 343 peptide in calmodulin is noticeably different in ground and excited states, indicating that the linear response picture is not appropriate in this case. The major difference is a more prominent fast component of solvation in the ground-state solvation dynamics. The ground-state energy gap correlation function is conjectured to be the best representation of protein relaxation, while that for the excited state seems to be influenced by the coupling of the protein modes to the large dipole created by excitation.

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.Bacillus thuringiensis is an insect pathogen with an unusual but highly specific mode of action. During the sporulation cycle it lays down a parasporal protein crystal which is rendered toxic on ingestion by susceptible insect larvae. The major component of crystals toxic to lepidoptera is a protein (protoxin) with a molecular mass of approximately 130 kDa [l -31. Treatment with thiol reagents at basic pH solubilizes the protoxin by cleaving the disulfide bonds which stabilize the crystal. Incubation of the solubilized protoxin with proteolytic enzymes or insect gut juice produces a 58 -70-kDa proteinaseresistant toxin derived from the N-terminal portion of the molecule [4,5]. The toxin then binds to receptors in the midgut epithelium, causing cell lysis and eventual larval death [6 -81. The details of the lytic mechanism are not yet established but it appears that the toxin generates small pores or localized perturbations in the plasma membrane, causing disruption of homeostatic ion regulation [9].Large proteins are generally organized into distinct structural units referred to as domains or subdomains, but the criteria used for this classification is somewhat subjective. It is clear that the protoxin is divided into at least two major domains: the carboxyl half of the molecule which is readily attacked by proteinases, and the toxin derived from the Nterminal half which is proteinase-resistant. The toxins from

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.

In membrane proteins, the extent to which polarity, hydrogen bonding, and van der Waals packing interactions of the buried, internal residues direct protein folding and association of transmembrane segments is poorly understood. The energetics associated with these various interactions should differ substantially between membrane versus water-soluble proteins. To help evaluate these energetics, we have altered a water-soluble, two-stranded coiled-coil peptide to render its sequence soluble in membranes. The membrane-soluble peptide associates in a monomer-dimer-trimer equilibrium, in which the trimer predominates at the highest peptide/detergent ratios. The oligomers are stabilized by a buried Asn side chain. Mutation of this Asn to Val essentially eliminates oligomerization of the membrane-soluble peptide. Thus, within a membrane-like environment, interactions involving a polar Asn side chain provide a strong thermodynamic driving force for membrane helix association.

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.

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We are developing all-synthetic model cofactor-protein complexes in order to define the parameters controlling non-natural cofactor activity. The long-term objective is to establish the theoretical and practical basis for designing novel enzymes. A non-heme pentadentate ligand (N4Py) is being developed as a template for the site-specific attachment of a designed four-helix bundle. Previously, we attached two unprotected peptides via CH(2)Cl handles to N4Py. In the presence of hydrogen peroxide, the iron(II) complex of this ligand (2a) generates an Fe(III)OOH intermediate (3a) that can oxidize a wide variety of organic compounds. Here, we describe the synthesis of 27, a N4Py derivative in which four three-carbon spacers have been introduced, and show that four copies of an unprotected, single-cysteine peptide can be coupled via a thioether linkage to the ligand. In addition, a divergent synthesis route to tetrabromide ligand 1b has also been developed, providing the opportunity to prepare alternative pentadentate ligands efficiently by four cross-coupling reactions on a single molecule. Also, two of the four bromides of 1b can be selectively addressed by magnesium-bromide exchange.