Glycine | EPFL Graph Search

Glycine (symbol Gly or G; ˈɡlaɪsiːn) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid (carbamic acid is unstable), with the chemical formula NH2‐CH2‐COOH. Glycine is one of the proteinogenic amino acids. It is encoded by all the codons starting with GG (GGU, GGC, GGA, GGG). Glycine is integral to the formation of alpha-helices in secondary protein structure due to its compact form. For the same reason, it is the most abundant amino acid in collagen triple-helices. Glycine is also an inhibitory neurotransmitter – interference with its release within the spinal cord (such as during a Clostridium tetani infection) can cause spastic paralysis due to uninhibited muscle contraction. It is the only achiral proteinogenic amino acid. It can fit into hydrophilic or hydrophobic environments, due to its minimal side chain of only one hydrogen atom. History and etymology Glycine was discovered in 1820 by French chemist Henri Braconn

Characterization of the 5HT3 receptor in live cells and in vitro

Veronica Ponce de Leon

The serotonin type 3 receptor (5HT3R) is a homopentameric, cation selective channel that mediates fast excitatory transmission in the nervous system. It is a member of the Cys-loop family of receptors that include the nicotinic acetylcholine receptor (nAChR), the γ-amino butyric acid (GABA) type A and C and glycine receptors. The 5HT3 receptor is involved in behavioral and digestive disorders and it is therefore an important therapeutic target. The 5-HT3 receptor was expressed in mammalian HEK cells (chapter 2) stably and transiently, and its expression was optimized to obtain 1.6 x 106 receptors per cell for its solubilisation (chapter 3) and purification (chapter 4). Solubilisation of the receptor was evaluated under different conditions such as using CHAPS and C12E9 detergents and homogenization procedures such as vortexing and thurraxing the membranes. Radioligand binding assays indicated that the amount of solubilised receptor was similar in both cases. The stability of the receptor at different temperatures was evaluated. The receptor was more stable when kept at -20 °C compared to 4 or 37 °C. Purification of the 5-HT3 receptor from HEK cells was performed by tandem IMAC (Immobilized Metal ion Affinity chromatography)/IMAC purification and IMAC/streptactin flow column chromatography. The 5-HT3R was obtained 50% pure and a yield of 10 pmoles of receptor binding sites per gram of starting material (dry cells) was obtained. Purified receptor preparations were homogenous. Finally, the receptor was concentrated 5.5 times, which allowed the incorporation of the receptor in lipid vesicles for further studies. Detergent solubilised 5-HT3R was incorporated in lipid vesicles and the functionality of the receptor was assessed by studying calcium ion permeability of the channel using calcium ion sensitive fluorophores (chapter 5). The effect of the cAMP dependent kinase (PKA), on the 5-HT3R was investigated by measuring the KD of the radioactive ligand [3H]GR65630 to the receptor in cells where PKA was activated or inhibited (chapter 6). Finally a dose-response curve of the binding activity of the 5-HT3R upon activation of the PKA was assayed in both HEK and CHO cells (chapter 6). Radioligand binding assays indicated that upon activation of the PKA, there was a decrease of the KD of 30% indicating an increase in the receptor binding affinity to 5-HT upon activation of the PKA (chapter 6). A kinase and cytoskeleton binding protein PDLI1 that was co-purified with the receptor was found phosphorylated upon PK activation (chapter 6). As PDLI1-like proteins are adaptor proteins between kinases, cytoskeleton proteins and sometimes receptors, the latter may be involved in the kinase activation of the receptor. These results contribute to the optimization of the receptor expression, solubilisation and purification in mammalian cells for its incorporation in lipid vesicles and for further structural studies. In addition, this work gave new insights to the mechanisms of channel regulation by PKA phosphorylation at the cell membrane and the intermediate proteins that are involved.

EPFL2009

Microhydration of Biomolecules: Revealing the Native Structures by Cold Ion IR Spectroscopy

Viktoriia Aladinskaia, Andrei Zviagin

The native-like structures of protonated glycine and peptide Gly3H+ were elucidated using cold ion IR spectroscopy of these biomolecules hydrated by a controlled number of water molecules. The complexes were generated directly from an aqueous solution using gentle electrospray ionization. Already with a single retained water molecule, GlyH+ exhibits the native-like structure characterized by a lack of intramolecular hydrogen bonds. We use our spectra to calibrate the available data for the same complexes, which are produced by cryogenic condensation of water onto the gas-phase glycine. In some conformers of these complexes, GlyH+ adopts the native-like structure, while in the others, it remains “kinetically” trapped in the intrinsic state. Upon condensation of 4–5 water molecules, the embedded amino acid fully adopts its native-like structure. Similarly, condensation of one water molecule onto the tripeptide is insufficient to fully eliminate its kinetically trapped intrinsic states.

2021

Molecular Hydrogen Messengers Can Lead to Structural Infidelity: A Cautionary Tale of Protonated Glycine

Antoine Louis Claude Masson, Thomas Rizzo, Evan Rowland Williams

The effects of tagging protonated glycine with either He or with between 1 and 14 H2 molecules on the infrared photodissociation (IRPD) spectra and the ion structure was investigated. Differences in the IR spectra with either a single He atom or H2 molecule attached indicate that even a single H2 molecule can affect the frequencies of some vibrational bands of this simple ion. The protonation site is the preferred location of the tag with He and with up to two H2 molecules, but evidence for H2 attachment to the hydrogen atom of the uncharged carboxylic acid is observed for ions tagged with three or more H2 molecules. This results in a 55 cm-1 red shift in the carboxylic acid OH stretch, and evidence for some structural isomers where the hydrogen bond between the protonated nitrogen and the carbonyl oxygen is partially broken as a result H2 molecules attached to this site is observed. These results are supported by theory, which indicates that H2 molecules can effectively break this weak hydrogen bond with three or more H2 molecules. These results indicate that large spectral shifts as a result of H2 molecules attaching to sites remote from the charge can occur and affect stretching frequencies as a result of charge transfer, and that tagging with multiple H2 molecules can change the structure of the ion itself.

American Institute of Physics2015

Characterization of the 5HT3 receptor in live cells and in vitro

Veronica Ponce de Leon

EPFL2009