Measures of Cysteine Acidity in Relation to Rates of Disulfide Formation
Poster Number
6
Introduction/Abstract
Cysteine and cystine residues play a major role in protein structure and the mediation of reactive oxygen species. Several neurodegenerative misfolding diseases (NMD) are associated with disulfide bond scrambling, including Alzheimer’s disease, Parkinson’s disease, prion-related disorders, and amyotrophic lateral sclerosis. Thioredoxin is a protein which is central to many pathways involving disulfides in biological systems. The difference in acidities of two active site cysteine residues of thioredoxin enables the reduction of disulfide bonds in proteins and peptides. The exact role the interactions between local residues and the functionality of the active site cysteines is unclear. We have found that disulfide bonds are formed between small cysteine containing peptides in solution in the absence of enzymes at different rates. Trends in the relative acidity of peptides were found to be congruent with trends in the rate of disulfide bond formation.
Purpose
The protein thioredoxin vital to every known form of life. The difference in acidies of the two active site cysteines of thioredoxin is crucial to its function: the reduction of disulfide bonds. The exact role the interactions between local residues in the active site of thioredoxin are unclear. Our research explores the relationship between disulfide bonds formation between small cysteine containing peptides and trends in the relative acidity of peptides in the gas phase and solution phase, with the purpose of generating a deeper understanding of disulfide chemistry.
Method
Solution phase disulfide formation in a series of tri- and penta- cysteine/alanine peptides was monitored using LC/MS using a serine/alanine analog internal standard. Gas and solution phase acidity measurements were performed on a series of acetylated peptides: Ac-CAA, Ac-AAC, Ac-CAAA, and Ac-AAAC.
Microscale UV and potentiometric titration were used to measure solution phase acidities in 1:1 methanol:water. Relative gas phase acidities were measured through a series of bracketing experiments involving the dissociation of a proton bound dimer during tandem mass spectrometry analysis.
Gas phase, implicit solvent and hybrid implicit/explicit solvent computational models were explored with Hartree Fock and density functional theory.
Results
Solution phase acidities, as well as experimental relative gas phase acidities, show that acetylated N-terminal cysteine peptides are more acidic than the corresponding isomeric C-terminal peptides. Trends in the rate of disulfide bond formation are congruent with trends in the relative acidity. Gas phase, implicit solvent and hybrid implicit/explicit solvent models computational models are explored and Ab initio computational studies are congruent with trends in experimental acidies and suggest helical-like character exists in certain small peptides (4 residues).
Significance
This research generates insight into how peptide structure and properties influence the reactivity of cysteine residues. Through our current research, we hope to gain insight into undiscovered factors modulating disulfide bond formation and reduction.
Location
DeRosa University Center
Format
Poster Presentation
Poster Session
Afternoon
Measures of Cysteine Acidity in Relation to Rates of Disulfide Formation
DeRosa University Center
Cysteine and cystine residues play a major role in protein structure and the mediation of reactive oxygen species. Several neurodegenerative misfolding diseases (NMD) are associated with disulfide bond scrambling, including Alzheimer’s disease, Parkinson’s disease, prion-related disorders, and amyotrophic lateral sclerosis. Thioredoxin is a protein which is central to many pathways involving disulfides in biological systems. The difference in acidities of two active site cysteine residues of thioredoxin enables the reduction of disulfide bonds in proteins and peptides. The exact role the interactions between local residues and the functionality of the active site cysteines is unclear. We have found that disulfide bonds are formed between small cysteine containing peptides in solution in the absence of enzymes at different rates. Trends in the relative acidity of peptides were found to be congruent with trends in the rate of disulfide bond formation.
