NMR study of oligonucleotides containing base pair mismatches and a human growth hormone peptide for the determination of solution structures
Abstract
Formation of unusual basepairs in DNA for random mutations in DNA was proposed in the sixties. These mismatches arise due to errors in replication, and from deamination of the 5-methylcytosine. Our interest in studying mismatches and other oligonucleotides has been two fold. One is related to $\sp{31}$P chemical shifts and the backbone structure of oligonucleotides. We wanted to find out the significance of the dispersion of $\sp{31}$P chemical shifts in oligonucleotides. We wished to address whether this dispersion in $\sp{31}$P chemical shifts is related to global structural parameters of oligonucleotides like helix twist and whether we can prove the relationship between $\sp{31}$P chemical shifts and the backbone torsional angles epsilon and zeta. How does a mismatch affect $\sp{31}$P chemical shifts and the backbone torsional angle? The second interest is related to solving the three dimensional structure of these biopolymers by using NMR data (NOESY distances) and computer simulations. The mismatches were incorporated in the d(CGCGAATTCGCG)$\sb2$ sequence because the structure of this sequence has been solved by X-ray crystallography and 2D NMR. All of our mismatches are at position three from the 5$\sp\prime$ side of the chain. The mismatches we are studying by NMR are GT, GU, GG, GA, and AC. Our major study of these mismatches has been in the assignments of the protons resonances (Chapter 3) and the phosphorus resonances (Chapter 4) by 2D NMR. We have also tried to answer the question about the relationships between $\sp{31}$P chemical shifts and global parameters for DNA such as the helix twist (Chapter 4). We have made substantial progress in determination of J(H3$\sp\prime$-P) coupling constants by 2D NMR and also in determining the relationship between the $\sp{31}$P chemical shifts and the backbone torsional angles by using the mismatch dodecamer sequences and the tetradecamer sequences (Chapter 5). The 2D NMR data for the GG and GT mismatch have been used to determine three dimensional structures by using distance restrained molecular dynamics (Chapter 6). The second project involved studying a 28 residue synthetic peptide by NMR. The peptide is the N-terminal fragment of the human pituitary growth hormone (hGH) which is a protein of 191 amino acids. Medium length peptides have been subjected to conformational investigations by NMR in recent times. Our goal was to determine the structure of the N-terminus 28 amino acid residue human growth peptide in aqueous solution by using 2-dimensional NMR (Chapter 7).
Degree
Ph.D.
Advisors
Gorenstein, Purdue University.
Subject Area
Biochemistry
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