Title

PROTON AFFINITY OF SHORT OLIGOPEPTIDES CONTAINING NATURAL AND UNNATURAL AMINO ACIDS

Introduction

In many active proteins, pKa perturbations can be observed in buried active-site residues. One such pKa perturbation is observed for the Lysine residue in the acetoacetate decarboxylase family of enzymes. It is hypothesized that basic residues closer to the C-terminus have higher proton affinities due to the molecule’s conformation ability in stabilizing the charged group. Our group aims to study conformational effects on basic residues incorporated into polyalanine peptides. This study aims to determine the influence of position on the proton affinity (PA) of basic residues including Lysine (Lys), Ornithine (Orn), 2,4-diaminobutanoic acid (Dab), and 2,3-diaminopropionic acid (Dap).

Method

Conformational search was carried out by Merck molecular force field and AM1 optimization of Geometry optimization and frequency calculations were performed on 10 low energy conformers to calculate thermal enthalpy correction at 298 K at the B3LYP/6-311+G(d) level. Single point energy calculations were then performed at the B3LYP/6-311++G(2d,p) level. The PA of each peptide was calculated using an isodesmic reaction with the reference base ethylamine. Acetylated (Ac) peptides AcAlaLys, AcLysAla, AcAlaDap, and AcDapAla were synthesized using solid phase peptide synthesis techniques. Mass spectrometric measurements were performed using an electrospray tandem-quadrupole mass MS (Varian 320 LC-MS). Using the extended Cooks’ kinetic method, collision induced dissociation (CID) at different energies were carried out to determine PA.

Results

Theoretical determinations of proton affinity have been performed on C-terminal basic dipeptides, AcAlaLys, AcAlaOrn, AcAlaDab, and AcAlaDap. The PA values of 245.56, 243.53, 241.72 and 236.68 kcal mol-1 were obtained, respectively. Theoretical determinations of proton affinity for N-terminal basic dipeptides AcLysAla, AcOrnAla, AcDabAla, and AcDapAla were also carried out and the PA obtained to be 243.41, 241.12, 238.87, and 234.31 kcal mol-1, respectively. An interesting trend was observed where decreases in the sidechain length of the basic residue decreases in PA. The other interesting trend observed is that acetylated peptides containing basic probes placed at the C-terminus have a higher theoretical PA than peptides containing basic probes placed at the N-terminus. It has been determined experimentally by mass spectrometric measurements that AcAlaLys has a PA of 249.5 ±2.0 kcal mol-1 and AcLysAla has a PA of 241.5 ±1.9 kcal mol-1 agreeing with our theoretical calculations. AcAlaDap and AcDapAla have proton affinities of 236.7 and 237.5 kcal mol-1 which conflicts with our qualitative observations and theoretical calculations.

Significance

Studying the effects of intrinsic properties on PA deepens our understanding of enzyme catalytic activity. This research leads into the advancement of engineered enzymes.

Location

DeRosa University Center, Stockton campus, University of the Pacific

Format

Poster Presentation

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Mar 25th, 10:00 AM Mar 25th, 3:00 PM

PROTON AFFINITY OF SHORT OLIGOPEPTIDES CONTAINING NATURAL AND UNNATURAL AMINO ACIDS

DeRosa University Center, Stockton campus, University of the Pacific

In many active proteins, pKa perturbations can be observed in buried active-site residues. One such pKa perturbation is observed for the Lysine residue in the acetoacetate decarboxylase family of enzymes. It is hypothesized that basic residues closer to the C-terminus have higher proton affinities due to the molecule’s conformation ability in stabilizing the charged group. Our group aims to study conformational effects on basic residues incorporated into polyalanine peptides. This study aims to determine the influence of position on the proton affinity (PA) of basic residues including Lysine (Lys), Ornithine (Orn), 2,4-diaminobutanoic acid (Dab), and 2,3-diaminopropionic acid (Dap).