Thesis
Understanding structural preferences found within the unfolded state of proteins and peptides through a spectroscopic analysis on A₅W, ionized trilysine, and various dipeptides
Master of Science (M.S.), Drexel University
Jun 2012
DOI:
https://doi.org/10.17918/00009311
Abstract
The unfolded state of proteins and peptides has been described as a random polymer in which all of the amino acid residues can sample all of the sterically allowed conformations without any influence of near-neighbors. This description has been questioned by numerous studies that revealed structural biases in the unfolded state. One of the most prevalent conformations found in the unfolded state is poly-proline II (PPII). Alanine has been shown to exhibit the highest preference for PPII besides proline. The degree to which individual amino acids sample different conformations in the unfolded state has been subject to an intense debate. With the development of more reliable experimental methods for determining structural biases, explicit conformational ensembles for individual amino acids have been characterized. Attempts have been made to reproduce these results through computation methods such as molecular dynamics (MD) simulations, but many simulations have produced results that do not agree with experimentally obtained conformational distributions. Recently, AMBER force fields have been re-parameterized in order to reproduce experimentally obtained nuclear magnetic resonance coupling constants of a short alanine-based peptide. In order to check the validity of the conformational distribution obtained from these MD simulations, we investigated the peptide H-A₅W-OH using electronic circular dichroism, vibrational circular dichroism (VCD), infrared spectroscopy, and NMR J-coupling constants. Additionally, we obtained end-to-end distances from Föster resonance energy transfer experiments. The distribution obtained from the MD simulations was used in a conformational distribution program in order to calculate the amide I' profiles, the J-coupling constants, and the end-to-end distance. This calculation produced an underestimated negative VCD couplet and an overestimated end-to-end distance compared to the experimental results. We desired to find a distribution that could reproduce all of the experimental data. To this end, all of the data was satisfactorily reproduced with a distribution that strongly biased polyproline II-like conformations. This reinforces previous studies that found alanine exhibits a high preference for polyproline II-like structures. Along with the intrinsic preferences described for amino acids residues, it has become evident that certain amino acids have the ability to modify the structural preferences of its neighbors. More specifically, lysine has been shown to modify alanine's preference towards more extended conformations over PPII. Also, the conformational ensembles of individual lysines appear to be at odds with the preferences observed for poly-lysines, which seems to be indicative of nearest neighbor effects. In order to shed some light on these discrepancies, we investigated ionized trilysine. Using a combination of vibrational and NMR spectroscopy, we determined conformational distributions for the central and N-terminal lysine residues. The central residue was found to predominantly sample conformations in the upper border of the upper left quadrant of the Ramachandran plot. This conformation was termed deformed poly-proline II (pPII^d). DFT calculations were performed on ionized trilysine and lysyllysyllysylglycine in vacuo. These calculations showed that pPII^d is stabilized by a rather strong hydrogen bond between the ammonium group of the central lysine and the carbonyl of the C-terminal residue. A similar analysis was carried out on triomithine, which is ornithine is a lower homolog of lysine. The conformational ensemble of this peptide was found to be very similar to trilysine, which suggests that the ammonium group of lysine's and ornithine's side chain is the chief determinant of these peptides' structure preferences. Much focus with regard to describing intrinsic conformational preferences in the unfolded state has been on alanine, but conformational ensembles of other amino acids have been proposed using various model systems. The dipeptide system has been shown to elicit differences in conformational ensembles among different amino acid residues. However, these results do not appear to agree with the results obtained using the unblocked tripeptide system. The third aim of this work seeks to shed light on these differences among the dipeptide and tripeptide systems.
Metrics
15 File views/ downloads
15 Record Views
Details
- Title
- Understanding structural preferences found within the unfolded state of proteins and peptides through a spectroscopic analysis on A₅W, ionized trilysine, and various dipeptides
- Creators
- Daniel J. Verbaro
- Contributors
- Reinhard Schweitzer-Stenner (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- vii, 98 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- College of Arts and Sciences; Chemistry; Drexel University
- Other Identifier
- 991021889004704721