Jake Bello

Studies on bovine pancreatic ribonuclease A and model compounds in aqueous 2-methyl-2,4-pentanediol: I. Amino acid solubility, thermal reversibility of ribonuclease A, and preferential hydration of ribonuclease A crystals

Abstract For X-ray investigations, RNase-A is crystallized from a mixture of 55% 2-methyl-2,4-pentanediol (MPD) and 45% water. Interactions of RNase-A and amino acids with this solvent have been studied. From the solubilities of amino acids in water and 55% MPD it is calculated that the unfolding of RNase-A in 55% MPD would have a positive free-energy change. RNase-A in 50% MPD and in water has nearly identical CD spectra. After being heated in 50% MPD and cooled, there is nearly complete reversibility of conformation, as indicated by CD, difference spectra, and enzymic activity. Cross-linked crystals of RNase-A are preferentially hydrated at up to 80% MPD (the limit of our studies).

Interaction of sodium dodecyl sulfate with tyrosyl chromophores in ribonuclease A and model compounds

Abstract Ultraviolet difference spectra, solvent perturbation difference spectra, and temperature perturbation difference spectra indicate that tyrosyl residues of model compounds are affected by sodium dodecyl sulfate. This effect is dependent on the nature of the model compound, being enhanced by positive charges, and is attributed to partial masking of the tyrosyl chromophores by sodium dodecyl sulfate. With reduced carboxymethylated ribonuclease as a model, all three difference spectral methods can be interpreted as indicating nearly complete externalization of tyrosyl chromophores in ribonuclease in the detergent. With small tyrosyl model compounds the calculated number of external tyrosyl residues depends on the nature of the model compound. Using net positively charged tyrosyl compounds as models, nearly 6 external tyrosyl residues are calculated for RNase. N-Acetyltyrosine amide or N-acetyltyrosine esters appear to be inadequate models for tyrosine in proteindetergent solutions because of their weak interactions with detergents.

Studies on bovine pancreatic ribonuclease A and model tyrosyl compounds in aqueous 2-methyl-2,4-pentanediol: II. Spectral investigation of salvation

Abstract Several spectroscopic techniques were used to study the states of solvation of the tyrosyls of bovine pancreatic ribonuclease A and tyrosyl model compounds in aqueous 2-methyl-2,4-pentanediol. The wavelength of the tyrosyl peak of the direct spectrum of RNase and the fluorescent quantum efficiency of RNase change little from 0 to 50% methylpentanediol concentration, while for model compounds these properties change markedly. Solvent perturbation difference spectra show very low apparent exposure of tyrosyl. Model compounds show pronounced “magic” mole fraction extrema in thermal perturbation difference spectra, while RNase shows a much smaller effect. The results are interpreted as indicating that the exposed tyrosyl residues of native ribonuclease A in aqueous methylpentanediol are preferentially hydrated relative to the model compounds.

Effects of aqueous alcohol and other non-electrolyte solutions on the thermal perturbation spectra of aromatic compounds

Difference spectrophotometry of identical solutions of aromatic compounds at different temperatures results in thermal perturbation difference spectra. These difference spectra were examined over the wavelength range 350-240 nm in water and in aqueous solutions of alcohols, ethylene glycol, dioxane, dimethyl sulphoxide, urea and tetramethylurea. The longest wavelength difference extremum has a positive ΔIµ/ΔT(change in molar extinction coefficient with temperature) which exhibits maxima at concentrations of alcohols similar to the maxima and minima shown by other properties of aqueous alcohol solutions. The more apolar the cosolvent the larger the value of ΔIµ/ΔT at the maximum and the smaller the mol fraction at which the maximum occurs. The results are attributed largely to changes in water-cosolvent interactions that are also observed by other physico-chemical methods.

Conformational changes in melittin upon complexation with an anionic melittin analog

Melittin and its Glu‐(7,21,22,23,24) analog upon mixing in equimolar concentrations form a hybrid oligomer with significant helical structure, in conditions in which each peptide separately adopts a largely disordered structure. The hybrid exhibits both cold‐ and heat‐induced denaturations similar to the phenomena exhibited by proteins. The hybrid also retains significant residual structure at higher temperature, similar to the ‘molten globular state’ that has been suggested for proteins. Melittin, at concentrations in which it forms helical tetramers, also exhibits these phenomena and may be used as a model for protein‐denaturation studies.