Tautomerism of Nucleic Acid Bases and PMR Studies of Histones. I. Proton Magnetic Resonance Studies of Tautomerism of Nucleic Acid Bases. II. Proton Magnetic Resonance Studies of Histones and a DNA-Histone Complex. III. Nuclear Magnetic Resonance Studies of Ribonuclease A-Inhibitor Interactions and Nucleotide Conformations

Citation

Lee, Chi-Yu Gregory (1972) Tautomerism of Nucleic Acid Bases and PMR Studies of Histones. I. Proton Magnetic Resonance Studies of Tautomerism of Nucleic Acid Bases. II. Proton Magnetic Resonance Studies of Histones and a DNA-Histone Complex. III. Nuclear Magnetic Resonance Studies of Ribonuclease A-Inhibitor Interactions and Nucleotide Conformations. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jtd2-ac90. https://resolver.caltech.edu/CaltechTHESIS:06132025-173756611

Abstract

PART I. Proton Magnetic Resonance Studies of Tautomerism of Nucleic Acid Bases

1. Cytosine. It has long been noted that the H₅ proton of cytosine and its related derivatives exhibits unusually broad resonances in the pmr spectrum under certain conditions of temperature and solution pD. We have examined this phenomenon as a function of temperature, concentration, and solution pD as well as the external magnetic field, and have shown that the linebroadening arises from chemical exchange between the amino and imino tautomers of the cytosine base. The observation of sharp H5 resonances in cytosine derivatives existing in only the amino or imino tautomeric structure supports this interpretation. This tautomeric exchange was found to be catalyzed by OD^- as well as the solvent molecules (D₂0). Analysis of the temperature and pD dependences of the H₅ linewidth for 5'-CMP led to the following rate law at 30°C:

- (d A/dt) _f = 1.2 x 10¹⁰ [OD^- [A] + 1.2 [D₂0] [A], (Msec^-1)

where [A] represents the major tautomeric species of 5'-CMP. Activation energies of 3.5 kcal/mole and 7.8 kcal/mole were obtained for the OD^- and D₂0 catalyzed steps respectively. The imino tautomer was estimated to be present to the extent of 15 ± 3% at room temperature in neutral aqueous solution.

2. Guanine. It has long been noted that the H3 proton of guanosine and its related derivatives exhibits unusually broad resonances in the pmr spectra under certain conditions of temperature and pD. We have examined this phenomenon as a function of temperature, concentration, and solution pD, as well as the external magnetic field, and have shown that the linebroadening arises from chemical exchange between the lactam and lactim tautomers of the guanine base. The observation of sharp H8 resonances in guanine derivatives where the guanine base is frozen in only the lactam and lactim tautomeric structure supports this interpretation. This tautomeric exchange was found to be catalyzed by OD^- and the solvent (D₂0). Analysis of the temperature and pD dependence of the H8 linewidth for 2'-GMP led to the following rate law at 30°c:

- (d A/dt) _f = 1.7 x 10⁹ [OD^- [A] + 0.7 [D₂0] [A], (Msec^-1

where A represents the major tautomeric species of 2'-GMP. Activation energies of 7 kcal/mole and 13 kcal/mole were deduced for the OD^- and D₂0 catalyzed steps respectively. The minor lactim tautomer was estimated to be present to the extent of 16 ± 3% at room temperature in neutral aqueous solution.

In most guanosine derivatives, with the notable exception of 2'-GMP, the Hg linewidth was also found to be strongly concentration dependent over the pD range 3 to 6. This concentration dependence has been interpreted in terms of the effect of intermolecular association on the lactam-lactim tautomeric equilibrium. Analysis of the data in terms of a rapid equilibrium involving the monomer, a hydrogen-bonded tetramer and stacked aggregates of this tetramer yielded a tetramer formation constant of(2.5 ± 0.5)x 10⁷ M^-3 and a tetramer stacking or association constant of 40 ± 10M^-1 in the case of 5'-GMP.

PART II. Proton Magnetic Resonance Studies of Histones and A DNA-Hostone Complex.

1. Solution Properties of Nucleoproteins - Histone I and Histone IIbI. The 220 MHz high resolution proton magnetic resonance (pmr) spectra of histone I and histone IIbl have been examined as a function of histone concentration, salt concentration as well as solution pD. The concentration studies of both histone I and histone IIbl revealed exclusively monomer-dimer equilibria in the presence or the absence of salt. The increase in the dimer formation of the proteins is attributed to the counter ion binding upon the addition of salt which results in shielding of the long range electrostatic repulsion and facilitating the hydrophobic interactions between the molecules. Changes in moles of the counter ion ΔV_x and water ΔV_w bound to each mole of histones upon the complex formation were determined experimentally. Temperature studies of histone I and histone IIbl indicate that in the presence of salt, the entropy change for the dimer formation of these proteins, AS becomes more positive than that in the absence of salt.

2 . Proton Magnetic Resonance Studies of the DNA-Histone I Complex. The DNA-histone I complex prepared by a continuous salt gradient dialysis was studied by high resolution proton magnetic resonance spectroscopy at various temperatures. It was concluded that about 15 to 20% of the side chain protons of the amino acid residues of the complexed histone I can be observed in the pmr spectra, and histone I in the complex is not removed from DNA when the latter is thermally denatured. The spectral behaviors of histone I when mixed directly with DNA or poly U were examined at various temperatures for the purpose of comparison. Possible structural models of this DNA-histone I reconstituted complex are presented on the basis of our experimental observations.

PART III. Nuclear Magnetic Resonance Studies of Ribonuclease A Inhibitor Interactions and Nucleotide Conformations.

1. A ³¹P NMR Study of Interactions of Ribonuclease A and its Inhibitor. The association between ribonuclease A and its product inhibitor uridine-3'-monophosphate has been studied by 3lp nmr spectroscopy. Analysis of chemical shifts and the linewidths of the ³¹ resonance of the inhibitor over a wide range of enzyme concentrations yielded the apparent dissociation rate constant 3200 ± 300 sec^-1 at 32°C for the enzyme-inhibitor complex and an activation energy of 3.5 ± 1.0 kcal/mole for this process. The chemical shift data suggest that the phosphate group of this inhibitor is simultaneously bound to two positively charged amino acid residues at the active site of the enzyme.

2. A Proton Magnetic Resonance Study of the Effects of pH on Nucleosides and Nucleotides in Aqueous Solution. Experimentally it was observed that ^JH₁ -H₂ of some guanine nucleosides and nucleotides varies with pD of the solution. This phenomenon was interpreted on the basis of the ring-base interactions due to the protonation of the guanine base at low pD's. From the results of the conformational analysis of these guanine derivatives, it was concluded that guanosine and 3'-GMP and 5'-GMP exist. predominantly in syn conformation when the guanine base is protonated at low pD's, while the anti conformation is still preferable at high pD's when the base is neutral.

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