Determination of the Structure of an Angiotensin Using NMR Spectroscopy
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NMR analysis is a complicated subject from both the fundamental physics as well as the spectral interpretation viewpoints. In the standard 1H-NMR examination, a sample is positioned in a robust magnetic field. The assimilation of electromagnetic radiation is calculated as diverse 1H nuclei are stimulated from their +1/2 turn states to their – 1/2 turn states, an approach known as resonance. The +1/2 nuclear spin situation is the lower energy spin state in which the 1H nuclear spin is aligned with the strong external magnetic field. The – 1/2 nuclear spin situations is the other “permitted” nuclear spin condition, and embodies the higher energy condition in which the nuclear turn is supported in opposition to the external magnetic field.
Angiotensin II is the active polypeptide hormone product present in rennin enzymatic cascade that responsible for sustaining vascular volume and blood pressure. Angiotensin regulates vasoconstriction and stimulation of adrenal aldosterone secretion. Angiotensin also controls the increased renal tubular absorption of sodium, increased cardiac contractility, and activation of the sympathetic nervous system. Added insight into the bioactive arrangement of Angiotensin II may be acquired from NMR spectroscopic researches of the hormone in selected surroundings. Because the local environment seen by Angiotensin II as it reaches the receptor-binding site may at some point bear a resemblance to a lipid milieu, a suspension of lipid vesicles in watery buffer must offer great solution for NMR researches of Angiotensin II.
Materials and Methods
This laboratory made use of aqueous models of Angiotensin II. This was dissolved in a peptide 1.1 mg in 500 micron liter of 50Mm 2HTris buffer. NMR samples that have lipid micelles were managed by dissolving 54mg of [38-2H] DodChoP in 500 micron liter of the [2H] Tris buffer.
All NMR measurement was established on a Bruker DMX 600 spectrometer at 30 deg Celsius. Two-dimensional NMR labs used for the conformational examinations of Angiotensin II encompassed nuclear Overhauser enhancement spectroscopy (NOESY) as well as total correlated spectroscopy (TOCSY). Nuclear Overhauser enhancement spectroscopy was obtained with mixing periods of 100 ms and 300 ms for the micelle model and 300 ms and 800 ms for the aqueous model. A mixing period of 50 ms was used for the TOCSY lab experiment. Two 2-dimensional spectra were obtained in the phase-sensitive mode, utilizing the method States-TPPI. Water suppression was obtained by utilized the technique Watergate. Phase-shifted sine-squared window operations were used along both dimensions before transformation.
The 1H-NMR chemical shift for Angiotensin II in aqueous solution were unambiguously recognized from cross peaks in TOCSY spectrum obtained for every sample 1H resemblances for the aqueous as well as micelle sample. Protons which showed great chemical shift movement (>0.1 ppm) upon shifting the peptide surrounding are illustrated in bold letters. NMR outcome for Angiotensin II in aqueous solution shows a one-dimensional proton spectrum obtained from Angiotensin II in buffer demonstrated two peaks signifying the existence of two conformations that are in gradual exchange. 1H-NMR spectra obtained for Angiotensin II demonstrated both minor and major groups of peaks. Relative intensities of line measured for two groups of peaks in the current study signify that the leading Trans conformer represents about 90% of the conformational ensemble.
Minor aliquots of a concentrated solution were added to the aqueous model of Angiotensin II and a single dimensional 1H-NMR spectrum was obtained after every addition. Dramatic line width widening was noticed for many of the amide protons including Ile NH, His NH, and Phe NH, along with a number of protons in the residue side chains such as His6C2H, His6C4H, Asp beta H, Asp alpha H, Pro7 sigma H, and Pro7 beta H. The reality that merely a few number of protons showed significant line widening in the presence of Myr2groPCho-SUV entails that a particular portion of Angiotensin II are impacted greatly compared to other Angiotensin II binding to the bilayer vesicles containing lipid. There were minor peaks from cis-trans isomerization of the peptide bond 1H spectrum obtained for Angiotensin II in the existence of DodChoP micelles. This outcome entails that in a solution containing phospholipid micelle solution, conformations of Angiotensin II containing a peptide bond cis, no longer make up a predominant part of the conformational ensemble. Reduction in structural flexibility because of the addition of micelles to the aqueous sample of Angiotensin II is most potentially accountable for spectral changes. Robust elements of Angiotensin II secondary arrangement were directly made obvious upon looking at the 100 ms NOESY spectrum taken for the micelle model. A massive number of medium-range as well as long range NOEs were noticed.
Angiotensin II adopts a spin-like arrangement in the peptide when placed in a constrained environment. It is also likely that the existence of a hydrophobic-hydrophilic interface offered by the mono- or bi- layer of the lipid is responsible for stabilizing the intramolecular interactions.

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