crystals Review Tryptophan, an Amino-Acid Endowed with Unique Properties and Its Many Roles in Membrane Proteins Sonia Khemaissa, Sandrine Sagan and Astrid Walrant * Sorbonne Université, École Normale Supérieure, PSL University, CNRS, Laboratoire des Biomolécules, LBM, 75005 Paris, France; [email protected] (S.K.); [email protected] (S.S.) * Correspondence: [email protected] Abstract: Tryptophan is an aromatic amino acid with unique physico-chemical properties. It is often encountered in membrane proteins, especially at the level of the water/bilayer interface. It plays a role in membrane protein stabilization, anchoring and orientation in lipid bilayers. It has a hydrophobic character but can also engage in many types of interactions, such as π–cation or hydrogen bonds. In this review, we give an overview of the role of tryptophan in membrane proteins and a more detailed description of the underlying noncovalent interactions it can engage in with membrane partners. Keywords: tryptophan; membrane proteins; noncovalent interactions 1. Introduction Citation: Khemaissa, S.; Sagan, S.; Among the naturally coded eukaryote amino acids, tryptophan (Trp) is unique in Walrant, A. Tryptophan, an terms of its physico-chemical properties. Considered as an aromatic residue similarly to Amino-Acid Endowed with Unique tyrosine (Tyr), phenylalanine (Phe) or histidine (His), it is however the sole amino acid Properties and Its Many Roles in that contains two rings in its lateral side-chain, namely the indole moiety composed of Membrane Proteins. Crystals 2021, 11, a benzene ring fused to a pyrrole ring, making it the largest coded amino acid in the 1032. https://doi.org/10.3390/ natural series. cryst11091032 Depending on the many different hydrophobic/hydrophilic scales that have been developed over the years by different research groups to rank amino acids, Trp is seen Academic Editors: Antonio Bauzá and Marta E. G. Mosquera as more or less hydrophobic. The nitrogen of its indole ring can indeed be engaged in hydrogen bonds, which may facilitate the solubility of proteins. Its large quadrupole allows Received: 27 July 2021 it to engage in intense π–π or π–cation interactions. In addition, Trp has a dipole moment Accepted: 24 August 2021 similar to Tyr but not Phe. All these specific physico-chemical properties make Trp unique Published: 27 August 2021 in biological functions and localizations of proteins. In this review, we develop how these unique Trp properties are crucial for membrane proteins and help to understand their Publisher’s Note: MDPI stays neutral membrane location and functions. with regard to jurisdictional claims in published maps and institutional affil- 2. Trp in Membrane Proteins iations. 2.1. Trp Localization at the Lipid/Water Interface of Membrane Proteins The analysis of amino acids distribution in proteins indicates that Trp is mostly located in transmembrane proteins, where it represents 3.3% of amino acid composition, whereas it only represents 1.2% for soluble proteins [1]. Copyright: © 2021 by the authors. In these transmembrane proteins, Trp has a strong preference for the bilayer interface Licensee MDPI, Basel, Switzerland. as demonstrated for the first time by Jacobs and White using neutron diffraction [2]. Since This article is an open access article then, other techniques have been used in order to determine Trp localization, including distributed under the terms and X-ray diffraction [3], nuclear magnetic resonance (NMR) [4], fluorescence spectroscopy [5] conditions of the Creative Commons and molecular simulations [6]. All these studies converge towards the same direction: Trp Attribution (CC BY) license (https:// is located at the lipid/water interface. Actually, Trp is not the only residue found at the creativecommons.org/licenses/by/ interface, aromatic residues in general have this preferential location in transmembrane 4.0/). Crystals 2021, 11, 1032. https://doi.org/10.3390/cryst11091032 https://www.mdpi.com/journal/crystals Crystals 2021, 11, x FOR PEER REVIEW 2 of 14 Crystals 2021, 11, 1032 2 of 13 Trp is located at the lipid/water interface. Actually, Trp is not the only residue found at the interface, aromatic residues in general have this preferential location in transmem- proteins in both α helix and β sheet structures, which is called “the aromatic belt” [7] brane proteins in both α helix and β sheet structures, which is called “the aromatic belt” (Figure1). [7] (Figure 1). FigureFigure 1. 1.Position Position of of amino amino acids acids in in transmembrane transmembrane proteins. proteins. Interestingly,Interestingly, aromatic aromatic residues residues are are not not randomly randomly distributed distributed at the at interfaces. the interfaces. Indeed, In- aromaticdeed, aromatic residues residues are often are mainly often mainly located locate at thed lipid–extracellularat the lipid–extracellular interface interface [8]. This [8]. observationThis observation is reported is reported in the in case the ofcase the of photosyntheticthe photosynthetic reaction reaction center, center, where where Trp Trp is mainlyis mainly located located in the in periplasmicthe periplasmic side [side1]. Another [1]. Another study study points points out this out particularity this particularity for 29 integralfor 29 integral proteins, proteins, highlighting highlighting the fact that the Trp fact is locatedthat Trp at is the located noncytoplasmic at the noncytoplasmic interface for αinterfacehelical transmembrane for α helical transmembrane proteins according proteins to a sequence-based according to a method.sequence-based The same method. study showsThe same that thisstudy residue shows is that more this present residue in α ishelical more present structures in thanα helical in β sheetstructures structures than [in7]. β However,sheet structures this tendency [7]. However, is not true this fortendency all proteins. is not Fortrue example, for all proteins. in the transmembrane For example, in segmentsthe transmembrane of human type segments I single-span of human membrane type I proteins, single-span Trp ismembrane found at both proteins, ends ofTrp the is hydrophobicfound at both domain, ends of whereas the hydrophobic Tyr is only domain, located whereas at the C-terminal Tyr is only boundary located at and the Phe C-ter- is foundminal inside boundary the hydrophobic and Phe is found domain inside and the at thehydrophobic Tyr position domain [8]. and at the Tyr position [8]. Trp is found at the interface and, more precisely, in the region of acyl carbonyl groups of theTrp lipid is bilayer,found at as the evidenced interface byand, the more chemical precisely, shift in change the region of the of signals acyl carbonyl attributed groups to theof cholinethe lipid group bilayer, in theas evidenced presence of by Trp the [ 9chem,10]. ical shift change of the signals attributed to the Anothercholine group study in shows the presence that Trp isof also Trp found [9,10]. in the glycerol region and in the hydropho- 1 2 bic coreAnother of lipid study bilayers, shows according that Trp is to alsoH andfoundH in NMR the glycerol studies. region The authorsand in the propose hydro- π thatphobic Trp locationcore of lipid in the bilayers, choline according region could to 1 beH and mainly 2H governedNMR studies. by cation– The authorsinteractions, propose whereasthat Trp inlocation the glycerol in the choline region, region other kindscould be of mainly interactions governed could by be cation– involved,π interactions, such as vanwhereas der Waals in the interactions, glycerol region, dipolar other interactions, kinds of interactions entropic contribution could be involved, or even such hydrogen as van bonding [10,11]. The interfacial localization of Trp allows it to establish interactions with der Waals interactions, dipolar interactions, entropic contribution or even hydrogen choline moieties, as mentioned above, but it can also be involved in interactions with other bonding [10,11]. The interfacial localization of Trp allows it to establish interactions with molecules present in its environment such as water with which hydrogen bonds are formed. choline moieties, as mentioned above, but it can also be involved in interactions with other Cationic residues in proteins, such as Arg and Lys, are located near phosphate moieties molecules present in its environment such as water with which hydrogen bonds are because a deeper localization inside a hydrophobic core is energetically unfavorable and formed. Cationic residues in proteins, such as Arg and Lys, are located near phosphate leads to specific lipid organization [12]. Trp can also interact favorably with these cationic moieties because a deeper localization inside a hydrophobic core is energetically unfavor- residues through cation–π interactions. able and leads to specific lipid organization [12]. Trp can also interact favorably with these Trp has a preference for the hydrophilic region over the hydrophobic core [13]. If cationic residues through cation–π interactions. we focus on its orientation in a bilayer, the benzene moiety of the indole prefers the Trp has a preference for the hydrophilic region over the hydrophobic core [13]. If we hydrophobic core, whereas the pyrrole moiety points towards the more hydrophilic part of focus on its orientation in a bilayer, the benzene moiety of the indole prefers the the lipid bilayer [8,14] Crystals 2021, 11, 1032 3 of 13 Molecular simulations for indoles in