Overview of Polar Amino Acids?

What are the polar amino acids?

Polar amino acids possess side chains capable of interacting with water owing to the presence of electronegative elements like oxygen, nitrogen, or sulfur. These atoms facilitate the formation of hydrogen bonds or dipole-dipole interactions among amino acids, making them hydrophilic. Serine (Ser, S) has a hydroxyl group (-OH) on its side chain. Threonine (Thr, T) comprises a hydroxyl group (-OH) and a methyl group. Tyrosine (Tyr, Y) has a phenolic hydroxyl group on an aromatic ring. Asparagine (Asn, N) has an amide group (-CONH₂) on its side chain. Glutamine (Gln, Q) has an extended amide group (-CONH₂). Cysteine (Cys, C) has a thiol group (-SH) on its side chain, capable of forming disulfide bonds. Histidine (His, H) has an imidazole group capable of engaging in hydrogen bonding. Polar amino acids are often located on the surface of proteins, facilitating interactions with the aqueous environment. Their side chains often engage in hydrogen bonding with other polar molecules, such as water. A multitude of polar amino acids participate in enzymatic activities owing to their capacity to establish connections with substrates or other molecules.

How to identify polar amino acids?

In order to identify polar amino acids, it is necessary to analyze the structure of the side chain (R-group) to ascertain whether it has the capacity to create hydrogen bonds or interact with water. Polar amino acids are hydrophilic and frequently engage in interactions or reactions with water or other polar molecules. Electronegative isotopes, such as oxygen, nitrogen, or sulfur, are frequently present in polar side chains. The side chain is capable of forming hydrogen bonds due to the dipoles that these atoms generate. It is probable that the side chain is polar and charged if it can either acquire or lose a proton at physiological pH (e.g., lysine, arginine, histidine, aspartate, glutamate). Nonpolar side chains are primarily composed of hydrocarbons (e.g., alkyl or aromatic groups without electronegative elements) and do not form significant hydrogen bonds (e.g., valine, leucine, phenylalanine).

Polar amino acids classification, structure and chart

Except for glycine, almost all α-amino acids have one asymmetric carbon atom, making them intrinsic to tautomerism. There is an L-form for every amino acid in a living thing. Amino acids can be classified as either nonpolar, polar uncharged, positively charged, or negatively charged according to their radical charges.

(1) Polar Uncharged Amino Acids

The amino acids with polar side chains that may form hydrogen bonds and possess no net charge at physiological pH are Serine, Threonine, Tyrosine, Cysteine, Asparagine, and Glutamine.

(2) Polar Charged Positively (Basic Amino Acids)

The amino acids with positively charged side chains at physiological pH are Lysine, Arginine, and Histidine. They comprise groups such as amines or imidazole that are protonated (positively charged).

(3) Polar Charged Negatively (Acidic Amino Acids)

These amino acids, such as glutamic acid (glutamate) and aspartic acid (aspartate), contain side chains that are negatively charged at physiological pH. They have deprotonated (negatively charged) carboxyl (–COO^–) groups.

Classification of amino acids by polarity and charge. (Dutta S., et al., 2020)

Amino acids are grouped as hydrophobic, hydrophilic, or polar vs. non-polar. (Frihart C R., et al., 2014)

Polar amino acids mnemonic

A mnemonic for remembering the polar amino acids (both charged and uncharged) is "Santa's Team Crafts New Quilts Yearly": S-Serine (Ser, S), T-Threonine (Thr, T), C-Cysteine (Cys, C), N-Asparagine (Asn, N), Q-Glutamine (Gln, Q), Y-Tyrosine (Tyr, Y). This mnemonic aids in remembering the polar uncharged amino acids, which are hydrophilic and capable of forming hydrogen bonds.

Positively Charged (Basic): "His Kingdom Rules", H-Histidine (His, H), K-Lysine (Lys, K), R-Arginine (Arg, R). Negatively Charged (Acidic): "Dragons Eat", D: Aspartate (Asp, D), E: Glutamate (Glu, E). These mnemonics may facilitate the learning of polar amino acids according to their charge and polarity.

Polar amino acids functions

(1) Alter interactions between transmembrane segments and detergents

Membrane proteins have α-helical transmembrane (TM) segments, which are predominantly composed of hydrophobic amino acids. These amino acids allow for the injection of water into the nonpolar membrane bilayer. Polar residues, on the other hand, are present in a significant number of these segments to fulfill either structural or functional purposes. The presence of these later residues hinders the local favorable acyl interactions that are necessary for solvation by hydrophobic media. Examples of such media include phospholipids in native bilayers and detergents that are utilized for in vitro characterization. We demonstrate that polar residues significantly alter the nature of the interaction between TM segments and the solvating detergent by using a series of Lys-tagged designed TM-like peptides (typified by KK-YAAAIAAIAWAIAAIAAAIAA-KKK). These peptides were designed in such a way that single-Asn residue substitutions (from Ile or Ala) were made successively from the center of the hydrophobic region toward the C-terminus. We were able to discover significant variations in the structures of the detergent–peptide complexes by using techniques such as tryptophan fluorescence, circular dichroism spectroscopy, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. These techniques allowed us to identify quite slight sequence alterations. As an example, the blue shift of the Trp fluorescence, which signifies the presence of local detergent solvation at this particular point, might vary by around 10 nanometers depending on the position of a single Asn substitution in a segment that is otherwise without any differences.

The side chain of an Asn residue typifies a strongly polar functional group that would be intrinsically unfavorable for hydrophobic interactions. When we placed the Asn residue in an otherwise hydrophobic sequence (characteristic of a protein TM segment), it is evident from the biophysical analysis performed that this residue may have a drastic effect on how the resulting segment interacts locally with the lipid environment of a detergent. Such large variation is observed even in model TM segments of identical composition, highlighting the nuances of such differences. While perhaps not considered classically amphipathic because of a lack of a truly hydrophilic surface, there is still a modest hydrophobic moment present in the TM helices studied here that dictates the relative conformation of these peptides within the detergent micelles. Specifically, if the net hydrophobicity is directed away from the Trp residue, as in the WT or variants in which the Asn is on the same surface of the helix, the Trp has a relatively small blue shift, indicating some exposure of this surface to water and/or the headgroup regions of the micelle. Conversely, a substitution opposite the Trp (such as I12N and A16N) causes further burial of this same helix surface in the center of the micelle. These findings support the notion of conformational sensitivity of a helix such as the present WT sequence that contains both a strongly hydrophobic surface and a moderately hydrophobic surface. Thus, in contrast to the situation in which a helix contains a hydrophobic face and a demonstrable hydrophilic face, one could term the WT sequence "lipopathic" because of the presence of two inherently hydrophobic faces with differing preferences to interact with lipid-like environments such as the core versus the surface of a detergent micelle.

There are two mechanisms in which polar residues change the solvation of SDS, even though their inclusion at most sites has no impact on membrane-based helicity. (i) Particles are smaller and the peptide gel migration rate is quicker, suggesting that detergent binding is lost, most likely in the region of the polar residue. Because of hydrophobicity, this effect is mostly seen in Ile-to-Asn substitutions, which vary in migration from the WT peptide more than Ala-to-Asn substitutions. Adding an Asn residue to spots near the Trp residue reduces the blue shift, suggesting that there is less detergent in those spots. ii) The Trp blue shifts show regular variability, which means that the distribution of SDS fluctuates. The helix takes on a kind of "interfacial" shape as a result of this event, which is connected to the lipopathy of the helix as more SDS is distributed to the surface that is hydrophobic. This circumstance permits the polar residue to be accommodated in an environment that is less hydrophobic, even when encircled by hydrophobic residues.

Schematic representation of detergent–peptide complexes. (Tulumello D V., et al., 2011)

(2) Regulate the functions of Na⁺/H⁺ exchangers

Membrane proteins that serve as sodium/potassium exchangers are commonplace. By exchanging intracellular H⁺ for external Na⁺, they control cytosolic pH in higher eukaryotic organisms. The Na⁺/H⁺ exchangers in yeast and Escherichia coli work in the reverse way, removing Na(+) from within the cell in return for H(+) outside the cell. The internal pH-sensitivity of Na⁺/H⁺ exchangers differs between antiporter types. Amino acids implicated in cation binding and transport, as well as pH sensitivity, have only lately been the subject of research. The ability to ionize within the physiological pH range makes histidine residues excellent candidates for H(+)-sensing amino acids. In the sodium/potassium exchangers NhaA from Escherichia coli and sod2 from yeast, histidine residues play a significant role in the enzyme's activity. It is possible that the pH-sensory region of NhaA is interacted with or regulated by His(225) of E. coli. His(367) is an important transporter in sod2 and could be a functional equivalent to NhaA's His(225). Although histidine residues are not essential for the mammalian Na⁺/H⁺exchanger to operate, a unique sequence rich in histidine at the C-terminal tail does affect activity to a lesser extent. Little is known about the other amino acids that play a role in cation binding and transport via Na⁺/H⁺ exchangers. The transport activity of NhaA and sod2 has been linked to polar side chain amino acids like glutamate and aspartate, although these acids have not been investigated in the mammalian Na⁺/H⁺ exchanger.

In mammalian cells, the NHE1 isoform of the Na⁺/H⁺ exchanger is a ubiquitous protein found in the plasma membrane that is responsible for regulating the pH of the intracellular environment. A site-specific mutagenesis approach was used in order to investigate the functional significance of polar amino acid residues that are conserved and found in portions of the protein that are linked with the membrane. A total of seventeen mutant proteins were evaluated by the analysis of intracellular pH changes in cells that had been transfected in a stable manner but did not have an indigenous Na⁺/H⁺ exchanger. All of the mutant proteins were directed to the plasma membrane in the appropriate manner and were produced at levels that were comparable to one another. While the mutation of Glu391 resulted in just a modest loss in activity, the amino-acid residues Glu262 and Asp267 were essential to the function of the Na⁺/H⁺ exchanger. In the presence of sodium ions, the Glu262→Gln mutant was largely produced as a deglycosylated protein, exhibiting heightened susceptibility to trypsin treatment. The activity of the Na⁺/H⁺ exchanger was restored by replacing the mutant Glu262, Asp267, and Glu391 with alternative acidic residues. However, the activity of the Glu262→Asp mutant was not altered by the presence of Na⁺ or H⁺ ions, despite the fact that its affinity for Li⁺ was reduced. The findings lend credence to the theory that the side-chain oxygen atoms found in a select group of amino acids that are strategically located play a significant role in the functioning of the Na⁺/H⁺ exchanger. Furthermore, the acidic amino-acid residues located at positions 262, 267, and 391 on the protein are strong candidates for being engaged in the process of Na⁺ coordination.

Models of the secondary structure of Na⁺/H⁺ exchangers. (Wiebe C A., et al., 2001)

Topological model of transmembrane segments VI, VII, and of amino acids 387±410. (Murtazina R., et al., 2001)

(3) As a component of channel constriction in the nicotinic acetylcholine receptor

By analyzing the single-channel conductances of systematically altered Torpedo receptors that were produced in Xenopus oocytes, researchers were able to analyze the channel pore of the nicotinic acetylcholine receptor (AChR). The mutations mostly change the size and polarity of uncharged polar amino acid residues that are located in the acetylcholine receptor subunits that are situated between the cytoplasmic ring and the extracellular ring. Based on the results that were obtained, we have come to the conclusion that a ring of uncharged polar residues, which includes threonine 244 of the α-subunit (αT244), βS250, γT253, and δS258 (also known as the central ring), and the anionic intermediate ring, which are situated in close proximity to each other in the assumed α-helical configuration of the M2-containing transmembrane segment, collectively form a narrow channel constriction of short length, which is situated in close proximity to the cytoplasmic side of the membrane. In addition, the findings indicate that individual subunits, namely the γ-subunit, are positioned in an asymmetrical manner near the channel constriction.

The channel-forming region of the AChR subunits. (Imoto K., et al., 1991)

(4) Regulate functionality of membrane toxin TisB

Both a reduction in cellular activity and a cessation of growth are characteristics of the dormancy state of bacteria. As an example, the long-term resistance to antibiotics that is attributed to a proportion of latent cells, which are referred to as persisters, is an example of a helpful technique that may be used to survive harsh situations. Within the context of Escherichia coli, we explore the membrane toxin TisB, which consists of 29 amino acids and is derived from the chromosomal toxin-antitoxin system tisB/istR-1. In response to DNA damage, TisB depolarizes the inner membrane, which ultimately leads to the promotion of a stress-tolerant state of dormancy among a limited portion of the population. In this study, we investigate the significance of charged and polar amino acids by using a plasmid-based system for modest tisB expression and single amino acid changes. We have observed that the key amino acids lysine 12 and glutamine 19 are of significant relevance for the functioning of TisB. This is further corroborated for lysine 12 in the native environment after it has been treated with the antibiotic ciprofloxacin, which is known to cause DNA damage. Finally, the study employs a library-based strategy to test more TisB variants in greater throughput, and by doing so, we discover that TisB-mediated dormancy requires the presence of at least one positive charge at the C-terminus (either lysine 26 or 29).

Importance of single amino acids for TisB functionality. (Leinberger F H., et al., 2024)

(5) Regulate lipid binding in the large hydrophobic cavity of CD1d

Natural killer T (NKT) cell activation is regulated by the CD1d protein, a nonpolymorphic MHC class I-like protein that regulates the presentation of self-and foreign-lipid ligands, glycolipids, or phospholipids, which in turn triggers the secretion of different cytokines. The A′ pocket of CD1d is a huge hydrophobic lipid binding site that may identify hydrophobic ligand moieties, such lengthy fatty acyl chains. One study demonstrated that the tiny polar areas situated deep within the hydrophobic A′ pocket might be used to modulate lipid binding, even though most lipid-protein interactions depend on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins. The structure-activity relationship studies showed that adding an amide group to the long fatty acyl chain, at the right places, improved the CD1d recognition of the glycolipid ligands, which were a series of α-galactosyl ceramide (α-GalCer) derivatives with polar groups in the acyl chain. Based on the results of the MD simulations and the WaterMap analysis, the increased activities were attributed to the creation of a hydrogen bond network in the A′ pocket, which allowed the amide groups to engage with polar residues like Gln14 or Ser28. The apolar residues around the hydrophobic pocket served to strengthen the hydrogen bonds by acting as a shield. It is possible to identify certain regions of mCD1d's lipid binding pockets, where polar residues are concentrated, as "hot spots" that affect the protein-ligand affinity.

Polar amino acid influences lipid binding inside the extensive hydrophobic cavity of CD1d. (Inuki S., et al., 2016)

References

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9. Inuki S., et al., Isolated polar amino acid residues modulate lipid binding in the large hydrophobic cavity of CD1d, ACS chemical biology, 2016, 11(11): 3132-3139.