Structural features of the initial active intermediate formed during acid denaturation of bovine pancreatic ribonuclease-A, Ph.D. Thesis

Abstract

The present thesis deals with structural investigations of the initial products formed during acid denaturation of ribonuclease A. We have divided our observations into two sections. Section I The nature of the deamidation reaction that ribonuclease A undergoes in highly acidic solution (0.5 N HCl, 30°C) was studied, and a fully active monodeamidated derivative, ribonuclease A??, was isolated. The structural alterations in ribonuclease A due to acid denaturation and reversal to the native state by inhibitor binding were established in this section. Section II The structures of ribonuclease A?? and ribonuclease A were compared at higher temperature and low pH. It was shown that deamidation is the only reaction ribonuclease A undergoes initially in 0.5 N HCl at 30°C, in accord with earlier observations. Ribonuclease A??, the monodeamidated protein, was isolated from 10?hour deamidated ribonuclease A (ribonuclease A_DD, where A_DD stands for acid?denatured derivative) on an Amberlite XE?64 column using phosphate. The nature of the lag period in the deamidation of ribonuclease A was investigated. It appears to be partly due to the formation of ribonuclease A??, which is further resistant to deamidation, and partly due to the unique structure of acid?denatured ribonuclease A, since oxidised protein does not show any lag period during deamidation. Ribonuclease A, after undergoing acid denaturation (0.5 N HCl, 30°C), was shown to have altered conformations at neutral pH, 25°C, as studied by spectrophotometric titrations, fluorescence, and ORD/CD techniques. Spectrophotometric titration curves of ribonuclease A?? and ribonuclease A are almost identical, indicating equal numbers of exposed and buried tyrosines. However, ribonuclease A_DD has 4.5 exposed tyrosine residues. The irreversible ionisation of phenolic groups occurs about 0.3 pH units earlier in ribonuclease A_DD. The non?deamidated component isolated from ribonuclease A_DD titrates similarly to ribonuclease A. It is assumed that phosphate used in chromatographic fractionation induces a conformational change and restores the original titration behaviour of tyrosines. This was confirmed by equilibrating ribonuclease A_DD with 0.2 M phosphate, pH 6.15, and studying the spectrophotometric titration after dialysis. The binding of 3??CMP also restores the original ionisation behaviour of tyrosine residues in ribonuclease A_DD. Ribonuclease A_DD generates a difference spectrum against ribonuclease A at neutral pH, 25°C, characteristic of a denatured molecule. No such spectrum is obtained between ribonuclease A?? and ribonuclease A. Spectrophotometric titrations of derivatives isolated at different stages of acid denaturation show two distinct phases of buried tyrosine exposure:

one tyrosine exposed early (constant until 7 hours), one additional tyrosine exposed between 7–10 hours.

Correlation of exposed tyrosines with rate of deamidation does not show a linear relationship. Interestingly, major structural transition occurs during 7–10 hours when deamidation rate is lowest. The quantum yield of ribonuclease A?? and ribonuclease A is the same, indicating similar tyrosine environments. However, acid?denatured ribonuclease A samples exhibit higher quantum yield at neutral pH, 25°C, increasing in two phases similar to titration results. The highest quantum yield is observed in ribonuclease A_DD. Fluorimetric acid titration indicates an earlier transition in ribonuclease A_DD than in ribonuclease A or ribonuclease A??. The latter two show very similar behaviour. Ribonuclease A_DD displays a non?cooperative transition. Alkaline fluorimetric titration shows that ionisation of three tyrosines quenches 90% of fluorescence in ribonuclease A and ribonuclease A??, but only about 70% in ribonuclease A_DD. Fluorescence intensity of ribonuclease A_DD returns nearly to that of ribonuclease A when equilibrated with phosphate. Binding of 3??CMP quenches emission intensity of ribonuclease A_DD but has no effect on ribonuclease A or ribonuclease A??. Titration with 3??CMP restores the original quantum yield of ribonuclease A in ribonuclease A_DD. Renaturation of ribonuclease A_DD from 8 M urea decreases fluorescence significantly; thermal renaturation does not. ORD spectra of ribonuclease A and ribonuclease A?? are very similar, indicating grossly similar conformations. However, [?]??? of ribonuclease A_DD drops to 3600, suggesting some loss of secondary structure. UV?CD spectra of ribonuclease A and ribonuclease A?? are virtually identical. Ribonuclease A_DD derivatives show varied spectra. The 240?nm positive peak is absent in 9?hr and 10?hr deamidated samples and shifted in 1?, 4?, and 7?hr samples. Ribonuclease A_DD at pH 1.75 shows greater structural change than ribonuclease A??, which in turn changes more than ribonuclease A. At pH 9.5, ellipticity around 240 nm is similar for ribonuclease A_DD and ribonuclease A, though different at neutral pH. Binding of 2?? or 3??CMP abolishes conformational differences between ribonuclease A and ribonuclease A_DD. After dialysis, 240? and 275?nm peaks return to native ribonuclease A intensity. Carboxypeptidase A action shows the C?terminal region is inaccessible in ribonuclease A, ribonuclease A??, and ribonuclease A_DD at 25°C. Thermal unfolding studies with chymotrypsin indicate that ribonuclease A?? unfolds earlier than ribonuclease A. Ribonuclease A_DD has similar thermal stability to ribonuclease A??. In presence of 2??CMP at 60°C, ribonuclease A, ribonuclease A??, and ribonuclease A_DD exhibit similar chymotryptic susceptibility. Pepsin susceptibility at pH 1.8, 25°C shows:

ribonuclease A?? loses activity slower than ribonuclease A, buried tyrosine exposure is faster in ribonuclease A??, fewer peptide bonds are cleaved in A?? than in A, pepsinolysis of ribonuclease A?? is “all?or?none.”

Methionines of ribonuclease A?? show greater reactivity toward o?benzoquinone (OBQ) than those of ribonuclease A. OBQ?modified proteins show reduced fluorescence. OBQ?modified methionine derivative is almost non?fluorescent. Conclusion Acid?denatured ribonuclease A does not refold to its native state after acid removal. Instead, it forms altered conformations at neutral pH with:

increased exposure of buried tyrosines, reduced secondary structure, altered methionine orientation, increased proteolytic susceptibility.

Ribonuclease A_DD is the most altered form. All altered conformations revert fully to the native state upon inhibitor or ligand binding.