Nuclear Magnetic Resonance Investigations: Structure, Function, and Dynamics

Citation

Perkins, Thomas Gardner (1982) Nuclear Magnetic Resonance Investigations: Structure, Function, and Dynamics. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/23sy-qt80. https://resolver.caltech.edu/CaltechTHESIS:05162018-141617618

Abstract

PART I

Carbon-13 nuclear magnetic resonance (nmr) spectroscopy has been used to investigate the chemical shifts and spin-lattice relaxation times (T₁) of ¹³CO bound to two derivatives of protoheme IX. The chemical shift is a function of the nature of the ligand trans to the ¹³CO and of the solvent. T₁ measurements of the complex 1-methylimidazole-protoheme IX dimethyl ester-¹³CO reveal that Chemical Shift Anisotropy (CSA) is the dominant relaxation mechanism for the heme bound ¹³CO. The aniostropy of the chemical shift tensor, Δσ, for the ¹³CO was found to be 584 ± 132 ppm. The chemical shifts are compared with those obtained for ¹³CO bound to the monomeric hemoglobin from the marine annelid Glycera dibranchiata.

PART II

Carbon-13 nuclear magnetic resonance (nmr) spectroscopy has been used to reinvestigate the spin-lattice relaxation times (T₁) of ¹³CO bound to human hemoglobin (HbA) and sperm whale myoglobin. It has been found that the Chemical Shift Anisotropy (CSA) and Dipole-Dipole (D-D) relaxation mechanisms contribute to the observed T₁ for the protein-bound ¹³CO. This observation can explain the lack of an observable nuclear Overhauser effect (NOE) for ¹³CO bound to HbA. A reanalysis of the previously determined relaxation times indicates that Δσ = 194 ± 37 ppm and r_eff = 1.81 ± 0.02 Å for ¹³CO bound to HbA. The significance of these results in relation to the postulated nucleophilic base interaction between the distal residue His-E7 and the protein bound CO is also discussed.

PART III

The spin-lattice relaxation (T₁) times for ¹³CO bound to New Zealand white rabbit hemoglobin (HbR) and the monomeric hemoglobin from the marine annelid Glycera dibranchiata (Hb-II) have been investigated. It has been found that the anisotropies of the chemical shift tensor, Δσ, in each protein are vastly different. These results support the existence of a nucleophilic interaction between His-E7 and the heme-bound ¹³CO in HbR. In addition, the geometry and rate of internal motion for ¹³CO bound to HbR have also been obtained.

PART IV

The pH dependence of the carbon-13 nuclear magnetic resonance (nmr) chemical shift for the C-2 carbon of selectively carbon-13 enriched histidine biosynthetically incorporated into the catalytic triad of the serine protease, α-lytic protease, has been reinvestigated at three magnetic fields. The spectra acquired at all fields yield a value for ¹J_13C-H at pH ~ 5 which is consistent with full protonation of the active site imidazole ring of His⁵⁷ at this pH. Hence, the catalytically important ionization of pKa ~ 6.7 can be assigned to His⁵⁷. At 125.76 MHz and pH ≾ 5, the carbon-13 spectrum of the enriched enzyme reveals two other structural forms of the histidine side chain within the protein which are not observed at lower fields. The presence of these species can explain previous carbon-13 nmr results which yielded an abnormally low pKa value for the catalytic histidine.

PART V

A general method is presented for obtaining the exchange rates for chemical systems undergoing slow exchange on the nuclear magnetic resonance (nmr) timescale. As an example of the generality of the method, ³¹P nmr spectroscopy has been used to measure the rate of exchange for the system [chemical equation included in scanned thesis' Abstract, p. xvii]. The exchange rates obtained with this method are compared to those measured using lineshape analysis.

PART VI

The binding of (R,S) d₅-ethanol (CD₃CHDOD) to horse liver alcohol dehydrogenase (LADH) has been studied using 500.13 MHz ¹H nuclear magnetic resonance (nmr) spectroscopy. In the presence of reduced nicotinamide adenine dinucleotide (NADH) the C-1 proton resonance moves such that the extrapolated chemical shift for the C-1 proton of the ethanol bound to the enzyme-coenzyme complex is shifted 0.82 ± 0.08 ppm upfield from the free ethanol resonance. The chemical shifts for the (R) and (S) hydrogens of the bound ethanol do not differ by more than 0.16 ppm.

PART VII

The structure of a formaldehyde-crosslinked dimer of verapamil, a Ca⁺² channel blocker, has been determined using 500.3 MHz ¹H nuclear magnetic resonance (nmr) spectra. The structure has been found to be asymmetric and, as with monomeric verapamil, possess a rigid conformation.

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