Effects of Hydrogen Bonding and Temperature Upon the Near Ultraviolet Circular Dichroism and Absorption Spectra of Tyrosine and O-Methyl Tyrosine Derivatives

Abstract

Abstract To gain information about the properties of tyrosyl residues buried within proteins, the circular dichroism (CD) and absorption spectra of tyrosine derivatives have been investigated in nonpolar solvents. N-Stearyl-l-tyrosine n-hexyl ester dissolved in methylcyclohexane (O—O band at 283 nm) was used to measure the effects of hydrogen-bonding agents. Adding low concentrations of dioxane, N,N-dimethylacetamide, 1-butanol, or methanol causes a 1- to 4-nm red shift in the absorption spectrum and a 10 to 25% increase in the dipole strength. The results with N,N-dimethylacetamide suggest that a hydrogen bond between a tyrosyl hydroxy group and a carbonyl oxygen of the peptide backbone may be one mechanism for producing a large red shift in proteins. CD spectra were recorded after the hydroxy group of N-stearyl-l-tyrosine n-hexyl ester had been hydrogen bonded. Dioxane and N,N-dimethylacetamide cause the CD spectra to red shift and intensify to the same extent as do the absorption spectra. Evidently hydrogen bonding to these compounds does not alter the conformation of this tyrosine derivative. In contrast, hydrogen bonding of N-stearyl-l-tyrosine n-hexyl ester to butanol or methanol causes a 50% loss of rotatory strength, suggesting an altered conformation. The dependence upon alcohol concentration is the same for both the CD and absorption alterations (halfmaximal effect at 30 mm alcohol). Evidence is presented that a polymeric form of the alcohol may simultaneously hydrogen bond to both the hydroxy group and the amide oxygen atom of N-stearyl-l-tyrosine n-hexyl ester. At concentrations greater than 15 µm, N-stearyl-l-tyrosine n-hexyl ester is partially aggregated in methylcyclohexane, causing changes in the CD and absorption spectra. The aggregate is proposed to be mainly a dimer in which the hydroxy group of each tyrosine residue is hydrogen bonded to the amide oxygen of the other residue (K ≈ 1000 m-1). To minimize aggregation, N-acetyl-O-methyl-l-tyrosine ethyl ester (N-Ac-O-Me-l-Tyr ethyl ester) and N-stearyl-Omethyl-l-tyrosine n-hexyl ester were studied. O-Methyl tyrosine derivatives have the same vibronic structure as does tyrosine, but the wave length position is not much shifted by polar organic solvents (O—O band at 283.5 ± 0.5 nm). The rotatory strengths of O-methyl-l-tyrosine derivatives are nearly identical with those of l-tyrosine derivatives in corresponding solvents. For N-Ac-O-Me-l-Tyr ethyl ester dissolved in methylcyclohexane at 297 K, the rotatory strength is especially large (1.3 x 10-40 c.g.s. with Δe = 0.75 m-1 cm-1 at 277 nm). Upon cooling N-Ac-O-Me-l-Tyr ethyl ester (dissolved in ether-isopentane-ethanol solvent) from 297 to 140 K, the negative rotational strength is intensified 10-fold. These results are interpreted in terms of temperature shifting the equilibrium distribution of the various conformers of N-Ac-O-Me-l-Tyr ethyl ester. The variable temperature CD technique may provide a way to detect motility of the tyrosyl side chains in proteins.