Biochimica et Biophysica Acta 1768 (2007) 2011–2025 www.elsevier.com/locate/bbamem Review The gramicidin ion channel: A model membrane protein ⁎ Devaki A. Kelkar 1, Amitabha Chattopadhyay Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Received 10 August 2006; received in revised form 9 May 2007; accepted 10 May 2007 Available online 18 May 2007 Abstract The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid–protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media. © 2007 Elsevier B.V. All rights reserved. Keywords: Gramicidin; Ion Channel; Tryptophan; Membrane Interface; Lipid–protein interaction Contents 1. Introduction..............................................................2012 2. Gramicidin conformation in membranes ...............................................2012 3. Gramicidin tryptophans at the membrane interface ..........................................2014 4. Synergestic approaches can reveal structure–function relationships .................................2014 5. Tryptophans in ion channel function .................................................2015 6. Tryptophan orientations in channel activity..............................................2017 7. Lipid–protein interactions influence gramicidin conformation and function . ............................2017 8. Membrane deformation and ion channel function: gramicidin as a molecular force transducer ...................2019 9. Gramicidin is a model membrane protein and ion channel ......................................2019 10. Conclusions and future perspectives .................................................2020 Acknowledgements .............................................................2020 References .................................................................2020 Abbreviations: DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine; DOPC, 1,2 dioleoyl-sn-glycero-3- phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero-3-phosphoserine; GMO, glycerylmonoolein; LPC, lysophosphatidylcholine; REES, red edge excitation shift; SDS, sodium dodecyl sulfate ⁎ Corresponding author. Tel.: +91 40 2719 2578; fax: +91 40 2716 0311. E-mail address: [email protected] (A. Chattopadhyay). 1 Present address: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland OR 97239, USA. 0005-2736/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2007.05.011 2012 D.A. Kelkar, A. Chattopadhyay / Biochimica et Biophysica Acta 1768 (2007) 2011–2025 1. Introduction (in 1939) and its success in clinical settings stimulated research into the practical application of other drugs such as penicillin Ion channels are transmembrane proteins that regulate ionic [23,24]. Gramicidin is produced during sporulation shortly after permeability in cell membranes. They represent an important the production of another antibiotic, tyrocidine [25]. The class of molecules due to their ability to serve as key elements in concentration of gramicidin in the bacterial cell is known to signaling and sensing pathways and to connect the inside of the reach up to 1 M during sporulation. Its function in B. brevis is cell to its outside in a selective fashion. They are crucial for not known, but it is believed to play a role in gene regulation normal functioning of cells and defective ion channels are during the shift from vegetative growth to sporulation and has implicated in a number of diseases collectively known as been shown to inhibit Escherichia coli RNA polymerase [25]. channelopathies [1,2]. Mutations in ion channels underlie Interestingly, the amino acid sequence of gramicidin consists diseases such as cystic fibrosis [3], renal disorders, certain of alternating L-andD-amino acids [16], in sharp contrast to types of hypertension, osteoporosis [4], and conditions such as most proteins which contain exclusively L-amino acids [26]. deafness [5] and reduced sleep [6]. The recent successes in Gramicidin is synthesized by non-ribosomal multienzyme crystallographic analyses of ion channels starting with the KcsA complexes, and the conversion of L-toD- amino acids takes potassium channel [7], the chloride channel [8] and more place during the biosynthesis. As mentioned earlier, the natural recently the voltage gated potassium channel [9] have provided mixture of gramicidins contains predominantly gramicidin A exciting molecular insights to ion channel function and structure (85%). The other peptides, gramicidin B and C differ in the [10]. However, it is becoming increasingly clear that static nature of the aromatic residue at position 11, where Trp (in A) is crystallographic structures of membrane proteins may not replaced by Phe and Tyr, respectively. Further, Val at position 1 always provide accurate representations of channel function is replaced by Ile in about 5 to 20% molecules. In spite of the [11–13]. Due to this ambiguity in the structural analysis of ion alternating sequence of L–D chirality generally not encountered channel function, simple models of ion channels continue to in naturally occurring peptides and proteins, gramicidin provide useful information to understand and characterize more represents a useful model for realistic determination of confor- complex systems [14]. mational preference of proteins in a membrane environment. The linear gramicidins are a family of prototypical channel This is due to the fact that the dihedral angle combinations formers that have been extensively used to study organization, generated in the conformation space by various gramicidin dynamics and function of membrane-spanning channels conformations are allowed according to the Ramachandran plot [14,15]. They are linear pentadecapeptide antibiotics with a [27]. The focus of this review is to highlight the organization molecular weight of ∼1900. Gramicidins are produced by the and dynamics of the gramicidin ion channel in membranes and soil bacterium Bacillus brevis, and consist of alternating L- and its suitability as a model membrane protein. It should be D-amino acids [16]. They form well-defined cation-selective ion mentioned here that this review does not cover in detail the channels in model membranes [17] with conductance of the significant advances made in recent years on the molecular order of ∼107 ions per second. Due to their small size, ready modeling of gramicidin conformation and free energy profiles availability and the relative ease with which chemical or in continuum electrostatics. These issues are covered in great modifications can be performed, gramicidins serve as excellent detail in several recent reports [14,28–31]. models for transmembrane channels. The natural mixture of gramicidins, often denoted as gramicidin A′ [termed gramicidin 2. Gramicidin conformation in membranes D (after René Dubos, who originally discovered gramicidin) in older literature], consists of ∼85% of gramicidin A, which has The unique sequence of alternating L- and D- chirality renders four tryptophan residues at positions 9, 11, 13, and 15 (see gramicidin sensitive to the environment in which it is placed. Fig. 1). Gramicidin A′ is readily available commercially and is Gramicidin therefore adopts a wide range of environment- fluorescent, due to the presence of tryptophan residues [18].It dependent conformations. Two major folding motifs have been has one of the most hydrophobic sequences known [19] and has identified for gramicidin in various media: (i) the single stranded very low solubility in aqueous solution (∼5×10− 7 M) [20]. helical dimer (the channel form) [32], and (ii) the double Gramicidins were first identified as an antibiotic produced by stranded intertwined helix (collectively known as the non- a culture of B. brevis isolated from soil samples by Dubos channel form) [33,34]. Interestingly, while double helical [21,22]. Gramicidin was the first antibiotic to be used clinically conformations are predominant in organic solvents, the conformation of the membrane-bound form of gramicidin is markedly different [35]. Ever since its initial isolation, a number of crystalline forms of gramicidin from a variety of organic solvents in the presence and absence of ions have been reported [36–41]. However gramicidin crystals have proved to be recalcitrant to X-ray analysis methods (possibly as a result of Fig. 1. Amino acid sequence of gramicidin A. Note that all amino acid side chains its intermediate size–too large for direct small molecule methods are either hydrophobic (Ala, Leu, Val) or amphipathic (Trp). In addition, the and too small for replacement methods traditionally used for -NH2 and -COOH termini are blocked making the sequence unusually hydrophobic. Alternating D-amino acid residues are underlined and the macromolecules) and rather limited structural data have been