<p>The work presented here employs cryogenic
ion spectroscopy for the study of protein structure, kinetics, and dynamics.
The main technique used is IR-UV double resonance spectroscopy. Here peptide
ions are generated through nano electrospray ionization, guided into a mass
spectrometer, mass selected, and then guided into a cryogenically held octupole
ion trap. Ions are subsequently cooled to their vibrational ground state
through collisions with 5 K helium allowing for high resolution IR and UV
spectra to be recorded. The IR spectra are highly sensitive to an ion’s
conformation, and the well resolved UV spectra provides a means generate
conformer specific IR spectra. With the use quantum mechanical calculations, it
is possible to calculate the vibrational spectra of candidate structures for
comparison with experimental spectra. Strong correlations between theory and
experiment allow for unambiguous structural assignments to be made.</p>
<p>
Structural studies are performed on β-turn motifs and well as salt-bridge geometries. Beta-turns are a commonly
occurring secondary structure in peptides and proteins. It is possible to
artificially encourage the formation of this secondary structural element
through the incorporation of the D-proline (<sup>D</sup>P) stereoisomer
followed by a gly or ala residue. Interestingly, the L-proline (<sup>L</sup>P) stereoisomer
is seen to discourage the formation of beta turn structure. Here were probe the
inherent conformational preferences of the diastereomeric peptide sequences YA<sup>L</sup>PAA
and YA<sup>D</sup>PAA. The findings agree with solution phase studies, the <sup>D</sup>P
sequence is observed to adopt a beta turn however, the <sup>L</sup>P sequence is
found to undergo a sterically driven <i>trans</i>
à
<i>cis</i> isomerization about the proline
amide bond. We find the energetics associated with this unfavorable interaction
and show the ability to reverse it by proper substitution of Ala<sub>2</sub>
for a Gly.</p>
<p>The studies directed towards gas
phase salt bridges have been limited to single amino acids or dipeptides.
Generally, these species are ionized using a metal ion or adducted with water
or excess electrons in order to stabilize a zwitterionic motif. Here we take
the first look at a salt bridge motif incorporated into polypeptide in order to
understand how the solvation from the secondary structure can aid in
stabilizing these motifs in non-polar environments. We find a unique salt
bridge motif in the YGRAR sequence in which the tyrosine OH acts as a neutral
bridge to form a network between the C-terminal arginine and the ion pair
formed between the central arginine and C-terminal carboxylate group. This
binding motif has not been discussed in literature and appears as an important
structural element in non-polar environments as all salt bridge character is
lost upon substituting Tyr for Phe. We are the process of mining the PDB for
these types of interactions. </p>
<p>To better understand how
cryo-cooling impacts the resulting population distribution at 10 K we measured
the distribution among the two major conformation of the YGPAA ion. This was
carried out using population transfer spectroscopy. In this method
conformational isomerization is induced vis single conformer infrared
excitation. The change in population can be related to the final population
distribution at 10 K. With this number, we were able to develop a cooling model
to simulate the change in the distribution as a function of cooling. The
cooling rates, were experimental established, and the isomerization rates and
starting population were theoretically derived through RRKM and thermodynamic
calculations. With these parameters and cooling model, we found that the room
temperature population distribution is largely preserved. When isomerization
events involve breaking a hydrogen bond, they become too slow to complete with the
cooling time scale of the experiment, effectively freezing in the room
temperature structures. These are important physical parameters to characterize
when performing structural studies at 10 K.</p>
<p>Finally, we demonstrate a 2-Color
IRMPD technique that is able to generate linear spectra at varied temperatures.
This is in sharp contrast to traditional IRMPD which results in non-linear and
skewed spectra. The importance of generating linear spectra when making
structural assignments is highlight by comparing the performance between both
techniques. Furthermore, with this technique we show the ability to record the
spectra of ion prepared with high internal energies. This provides
spectroscopic snapshots of the unfolding events leading to dissociation.
Overall, the versatility of this technique to record ground state spectra
comparable to IR-UV DR, to record linear spectra at room temperature, and to
probe dynamics proves this technique to be useful in the field of ion
spectroscopy.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/11327921 |
| Date | 06 December 2019 |
| Creators | Christopher P Harrilal (8082680) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/APPLICATION_OF_CRYOGENIC_INFRARED_AND_ULTRAVIOLET_SPECTROSCOPY_FOR_STRUCTURAL_AND_DYNAMIC_STUDIES_OF_GAS_PHASE_IONS/11327921 |
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