Crystallizing Transmembrane Peptides in Lipidic Mesophases
Nicole Ho ¨fer,†‡David Araga ˜o,†‡and Martin Caffrey†‡*
†MembraneStructuraland Functional BiologyGroup,Schoolof Biochemistry andImmunology,and SchoolofMedicine,Trinity College,Dublin,
Ireland; and‡Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland
lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not
apply to small protein targets having %4 transmembrane crossings. In our study, the hypothesis that the inherent flexibility of the
mesophase would enable crystallogenesis of small proteins was tested using a transmembrane pentadecapeptide, linear gram-
icidin, which produced structure-grade crystals. This result suggests that the in meso method should be considered as a viable
means for high-resolution structure determination of integral membrane peptides, many of which are predicted to be coded for in
the human genome.
Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in
Received for publication 17 February 2010 and in final form 3 May 2010.
The high-resolution structures of important membrane
proteins have been obtained with crystals generated by the
so-called in meso method (1). The method involves an
initial reconstitution of the target protein into the bilayer
of a cubic mesophase followed by the addition of a precipi-
tant that triggers nucleation and crystal growth (2). At its
simplest, the cubic phase consists of lipid and water. The
lipid exists as a continuous, highly curved bilayer that
divides the aqueous component into two interpenetrating
but noncontacting channels.
The inmeso method has been shown to be quitegeneral in
that it has been used to solve crystal structures of prokary-
otic and eukaryotic proteins—proteins that are monomeric,
homo- and heteromultimeric, chromophore-containing and
chromophore-free, and a-helical and b-barrel proteins. Its
most recent successes are the human, engineered b2-adren-
ergic and adenosine A2AG protein-coupled receptors (3).
A proposal has been advanced for how in meso crystallo-
genesis takes place at a molecular level ((1,4); and see our
Fig. 1). Typically, it begins with an isolated biological
membrane that is treated with detergent to solubilize the
target protein. The protein-detergent complex is purified by
reconstitution of the purified protein into the bilayer of the
cubic phase. The latter is bicontinuous in the sense that
both the aqueous and bilayer components are continuous in
three-dimensional space (Fig. 1). The protein retains its
native conformation and activity and is free to move within
the plane of the cubic phase bilayer. A precipitant is added
to the mesophase which triggers a phase separation. Under
conditions leading to crystallization, one of the separated
phases is lamellar and becomes enriched in protein. The
locally high concentration of protein (that may or may not
include native membrane lipid), in conjunction with an
appropriate bathing solution composition and bilayer micro-
structure, act to facilitate nucleation and crystal growth.
Aspects of this model are supported by experiment (1).
To be a generally applicable method it must work with all
sorts of membrane proteins and peptides, both large and
small. In a detailed theoretical analysis of the in meso
process, Grabe et al. (5) concluded that it will only work
with proteins having at least five transmembrane helices
and that it was not suitable for ‘‘small proteins’’. However,
giventhe inherent flexibilityof the compartments in a bicon-
tinuous mesophase, we speculated that the in meso method
would prove useful for a broad range of membrane protein
types and sizes. The in meso structures solved to date (see
www.mpdb.tcd.ie (6)) support this view. However, the latter
group does not include small proteins of the type referred to
by Grabe et al. (5). In this study, we set out to explore the
lower size limit of the method and chose to work with linear
gramicidin, a transmembrane pentadecapeptide.
Gramicidin is an antibiotic produced nonribosomally by
Bacillus brevis (7). It acts, in part, by creating pores in
membranes, rendering them incapable of supporting life-
sustaining transmembranal gradients. Naturally occurring
gramicidin is a mixture of isoforms: gA (80%), gB (6%),
and gC (14%). The amino acid sequence of gA is:
Formyl ? NH ? L ? Val ? Gly ? L ? Ala ? D ? Leu?
L ? Ala ? D ? Val ? L ? Val ? D ? Val ? L ? Trp?
D ? Leu ? L ? Trp11? D ? Leu ? L ? Trp ? D ? Leu
? L ? Trp ? CO ? NH ? CH2? CH2? OH:
In gB and gC, Trp at position 11 is replaced by L-Phe
and L-Tyr, respectively (8). The ion-conducting form of
Editor: Lukas K. Tamm.
? 2010 by the Biophysical Society
Biophysical Journal Volume 99 August 2010 L23–L25L23