Effective Acidities of Isomeric Asp-PolyPro Peptides in the Gas-Phase and in Solution

Lead Author Affiliation

Bioanalytical Chemistry, Physical Chemistry, Biochemistry

Lead Author Status

Doctoral Student

Fifth Author Affiliation

Chemistry

Fifth Author Status

Faculty

Research or Creativity Area

Natural Sciences

Abstract

Introduction

Acidity is an important parameter gauging biochemical properties. The effective acidity of ionizable amino acid residues is largely influenced by the position of the residue, the conformation of the peptide motif, and the local environment around the residue in proteins. Polyproline’s ability to switch between two unique helical conformations, PPI and PPII, in solution and in the gas-phase makes it an ideal model to study the effective acidities in different phases. This work focuses on comparing the acidities measured in the gas-phase and in various solvents for a series of aspartic acid-containing polyproline peptides. The results will reveal acidity trends of isomeric peptides, which will help to better understand the role of polyproline in biological systems.

Methods

The peptides were synthesized in-house using the solid phase peptide synthesis protocol. The solution phase acidity was determined by measuring the pKa values by potentiometric titrations with an 8220 BNWP ROSS micro pH electrode. Mixed solvents consisting of methanol and water with varying ratios were used in the measurements. The gas-phase acidities were measured using a tandem quadrupole mass spectrometer with an ESI source. The CID bracketing experiments and the extended Cooks kinetic method were employed to determine the qualitative and quantitative gas-phase acidities, respectively. The peptide conformations and the theoretical gas-phase acidities were obtained computationally by extensive conformational search followed by energetic calculations.

Preliminary Data

The acidities of a series of aspartic acid (D)-polyproline (P) peptides have been measured in both solution and in the gas-phase. The influence of three factors on the acidity of these peptides are determined, including the position of the aspartic acid (D) residue, the presence or absence of an acetyl group on the N-terminus, and the length of the peptide ranging from 3-9 amino acid residues. The peptides are amidated at the C-terminus, and therefore, the side-chain of the aspartic acid is the only acidic site for deprotonation. In the gas-phase, the acidity enhances as the aspartic acid residue is shifted from the N- to the C-terminus, the peptide chain is elongated, and the N-terminus is acetylated. The position of the aspartic acid residue has a significant effect on the acidity compared to other factors. For isomeric pentapeptides, the gas-phase acidity follows the trend Ac-P5D > P5D > Ac-P2DP2 > P2DP2 > Ac-DP5 > DP5. In solution, peptides with a C-terminal aspartic acid are more acidic (lower pKa values) than those with an N-terminal aspartic acid. However, peptides with aspartic acid in the middle are least acidic. This difference in acidity is larger in solvents containing higher fraction of methanol than water. In a solvent consisting of MeOH:H2O (1:1, v:v), the pKa values are Ac-P2D (5.14), Ac-P3D (5.06), Ac-P4D (5.09) and Ac-DP4 (5.13). In a solvent of MeOH:H2O (95:5, v:v), pKa values are Ac-P2D (6.32), Ac-P3D (6.36), Ac-P4D (6.31) and Ac-DP4 (6.45). The polarity of the solvent is likely to affect the peptide conformation.

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Effective Acidities of Isomeric Asp-PolyPro Peptides in the Gas-Phase and in Solution

Introduction

Acidity is an important parameter gauging biochemical properties. The effective acidity of ionizable amino acid residues is largely influenced by the position of the residue, the conformation of the peptide motif, and the local environment around the residue in proteins. Polyproline’s ability to switch between two unique helical conformations, PPI and PPII, in solution and in the gas-phase makes it an ideal model to study the effective acidities in different phases. This work focuses on comparing the acidities measured in the gas-phase and in various solvents for a series of aspartic acid-containing polyproline peptides. The results will reveal acidity trends of isomeric peptides, which will help to better understand the role of polyproline in biological systems.

Methods

The peptides were synthesized in-house using the solid phase peptide synthesis protocol. The solution phase acidity was determined by measuring the pKa values by potentiometric titrations with an 8220 BNWP ROSS micro pH electrode. Mixed solvents consisting of methanol and water with varying ratios were used in the measurements. The gas-phase acidities were measured using a tandem quadrupole mass spectrometer with an ESI source. The CID bracketing experiments and the extended Cooks kinetic method were employed to determine the qualitative and quantitative gas-phase acidities, respectively. The peptide conformations and the theoretical gas-phase acidities were obtained computationally by extensive conformational search followed by energetic calculations.

Preliminary Data

The acidities of a series of aspartic acid (D)-polyproline (P) peptides have been measured in both solution and in the gas-phase. The influence of three factors on the acidity of these peptides are determined, including the position of the aspartic acid (D) residue, the presence or absence of an acetyl group on the N-terminus, and the length of the peptide ranging from 3-9 amino acid residues. The peptides are amidated at the C-terminus, and therefore, the side-chain of the aspartic acid is the only acidic site for deprotonation. In the gas-phase, the acidity enhances as the aspartic acid residue is shifted from the N- to the C-terminus, the peptide chain is elongated, and the N-terminus is acetylated. The position of the aspartic acid residue has a significant effect on the acidity compared to other factors. For isomeric pentapeptides, the gas-phase acidity follows the trend Ac-P5D > P5D > Ac-P2DP2 > P2DP2 > Ac-DP5 > DP5. In solution, peptides with a C-terminal aspartic acid are more acidic (lower pKa values) than those with an N-terminal aspartic acid. However, peptides with aspartic acid in the middle are least acidic. This difference in acidity is larger in solvents containing higher fraction of methanol than water. In a solvent consisting of MeOH:H2O (1:1, v:v), the pKa values are Ac-P2D (5.14), Ac-P3D (5.06), Ac-P4D (5.09) and Ac-DP4 (5.13). In a solvent of MeOH:H2O (95:5, v:v), pKa values are Ac-P2D (6.32), Ac-P3D (6.36), Ac-P4D (6.31) and Ac-DP4 (6.45). The polarity of the solvent is likely to affect the peptide conformation.