No abstract

Melittin, a 26-residue peptide from bee venom, is transformed from a largely random to a largely alpha-helical conformation at elevated pH. At 3 x 10(-5) M melittin, circular dichroism spectra show a transition with a pK near 9.6. At 8 x 10(-5) M, two approximately equal transitions occur with pKs at 7.2 and 9.6. At 6 x 10(-4) M, a single transition is seen with a pK of 6.8, followed by a more gradual increase to at least pH 11. The transitions near pH 7 presumably arise from deprotonation of the alpha-amino group. When the amino groups are acetylated or succinylated, a 60% alpha-helical conformation is adopted at neutral or low pH. The acylated melittins form more stable oligomers than does native melittin.

The interactions of N-methylacetamide (NMA) and N,N-dimethylacetamide (DMA) with denaturants were studied by viscometry, calorimetry, and crystallography. Lithium and calcium chlorides show strong interactions by all three criteria; these cations probably interact with peptide groups.Alkali thiocyanate, nitrate, and perchlorate salts show T here are two general mechanisms by which salts may denature proteins: (1) interaction with the protein, probably at peptide or other polar groups (possibly including the ir electrons of the aromatic side chains);(2) alteration of the solvent structure. We shall not consider here salts with large apolar chains which can be bound to apolar regions of the protein. Geschwind (1960) reported that aqueous mixtures of urea and lithium bromide showed high viscosities; similar viscosity increases were found by Bello and Bello (1961,1962) for aqueous solutions of lithium chloride or bromide with N-methylacetamide (NMA)' or N,N-dimethylacetamide (DMA). DMA was investigated as an analog of proline and hydroxyproline which contain no NH group when present in peptide linkage. These observations led to the suggestion that lithium halides interact with the amide groups, and that they denature proteins by interaction with peptide or other polar groups. Crystalline complexes of NMA with lithium chloride and bromide were obtained (Bello and Bello, 1962) from an aqueous environment and their structures have been determined by X-ray diffraction (Haas, 1964). In these complexes there are carbonyl-lithium interactions and "-halide hydrogen bonds. In this communication, we report viscometric, calorimetric, and crystallographic data on the interactions of NMA and DMA with a number of denaturing salts.

1299are the stoichiometric coefficients.of the ions upon ionization, then the activity of the electrolyte as a whole is where r = r1 + r2 and f, the mean activity coefficient, takes into account the possible deviation from free volume ideality. If the free volume fractions are now written in terms of the molality of the hydrated solute and if the average volume ratio of the electrolyte as a whole, (qsl + rZsz)/r, is denoted by s, then cy12 is given by a = alrz a2ra = f' alri o 1 2 r~ = f ' a i z (13) If one now applies (3), cancels all common terms, substitutes (l), and makes use of the same technique which led to ( 5 ) , one obtains the desired equationThere are some interesting special cases. If s = 1, mole fractions result, and (15) reduces For the ideal non-electrolytes, r and f' are both to (5).unity and (15) becomes( 1 6 ) If there is no solvation, (16) reduces to equation 9.21 of reference 4. Equation 16 has been fitted to the data for aqueous sucrose from 0.1 to 5m with h = 8.7, 'z = 2.7. The fit is about as good as that of 9.214 with h = 0, z = 5. If z were really proportional to the molal volume, it would have been larger than h. If the Debye-Hiickel equation is applied to the solvated ions as before, one obtains for aqueous solutions z+ z-A 4 1 + a~& + 2.303[1 -0.018(h -rs)m] logy = log [I -0.018(h -rs)m] Since a t present there is no a priori way to determine s, equation 17 remains a three parameter equation. Acknowledgment.-The author wishes to acknowledge conversations with Professor Joseph E. Mayer and Dr. Edward V. Sayre. He is indebted to Miriam Miller, Lorraine Fischer and Stuart Rideout for very generous aid with the computations.O.O18s(s -1)rm (17) The effects of a series of salts and acids on the melting points of gelatin gels have been studied. The effects of ions, of the same or opposite charge, are additive. There is a correlation between the binding of ions, as indicated by pH changes, and their effects 011 the melting point. However, by the use of amino-acetylated gelatin and a guanidino-nitrated, hydroxylsulfated gelatin, it is shown that binding of anions at amino, guanidino or hydroxyl groups is not responsible for melting point changes. B y the use of carboxyl-esterified gelatin and hydroxyl-acetylated gelatin, it is shown that binding of cations at carboxyl or hydroxyl groups is not the cause d melting point reduction. Iron (111) ion, at low concentration, raises the melting point by inter-or intramolecular cross-linking through the carboxyl groups, I n agreement with previous reports polarizable anions are effective melting point reducers with diiodosalicylate being the most potent observed.

Melittin (MLT), a 26-residue cationic (net charge +5 at pH 7.2) peptide from bee venom, is well known to be a monomeric, approximately random coil; but when its charges are reduced by titration, by acetylation (net charge +2) or succinylation (net charge -2), or by screening by salt, it goes over to tetrameric alpha-helix. The conversion is promoted by raising the peptide concentration. The tetramer is held together by hydrophobic forces. We have changed the net charge to -6 by acylation with acetylcitric anhydride (a new acylating agent); this anionic derivative forms tetrameric helix at neutral pH, without salt, and at relatively low concentration, conditions under which the cationic MLT does not become helical. Thus, a high net charge is not sufficient to prevent association and helix formation. We have synthesized an anionic melittin analogue of MLT (E-MLT; net charge -4) in which all five lysine and arginine residues are replaced with glutamate, and acetyl and succinyl derivatives of E-MLT (net charges -5 and -6). All three of these are resistant to helix formation. They require much higher NaCl or NaClO4 concentration for helix formation than does MLT. Even CaCl2, MgCl2, and spermine.4HCl are less effective in helicizing E-MLT than MLT. MLT, at pH 7.2, shows increasing helix as the peptide concentration increases (8-120 microM), but E-MLT and its acyl derivatives do not. MLT and acylated MLTs in the helical tetramer show both cold- and heat-induced unfolding, with maximum stability near room temperature. At high temperature, a significant amount of residual structure remains. Heating (to 100 degrees C) monomeric MLT (i.e., MLT at low concentration) or E-MLT results in a monotonic increase in negative ellipticity. In 1.0 M NaCl, E-MLT (at sufficiently high concentration) also shows cold and hot unfolding. The results are discussed in respect to charge-charge and charge-dipole interactions, and hydrophobic effects. E-MLT is also discussed in relation to proteins of halophilic bacteria, which have higher proportions of anionic residues than do corresponding proteins of nonhalophiles.