Internal packing of helical membrane proteins

Abstract

Internal residues determine in large part the way proteins fold and function. Interiors of soluble proteins are tightly packed with densities approaching those of crystals of small organic molecules (1, 2). The stability of both native (3) and designed proteins (4) is closely correlated with the packing of core residues. Increased packing appears to be one mechanism by which the extremely stable hyperthermophilic proteins gain increased stability over their mesophilic counterparts (5). Packing analyses have been critical for evaluating structural models (6–8), designing novel proteins (4, 9), and generally understanding how the final tertiary structure of a protein is encoded in its primary sequence (2). The recent structure determinations of several large membrane protein complexes (10–20) provide a reasonable set of data to investigate the packing properties of helical integral membrane proteins.

The internal packing of membrane proteins is in many ways simpler than that of soluble proteins. The hydrophobic core of polytopic integral membrane proteins is most often formed by well-packed membrane spanning α-helices. Only in a few known cases does the transmembrane (TM) region consist of an antiparallel bundle of β-strands, as in the porins (21) or a combination of both helix and β-strands, as has been suggested for the acetylcholine receptor (22, 23). Helices spontaneously form on insertion of a hydrophobic sequence into a membrane bilayer because of the negative free energy associated with hydrogen bonding of the polar backbone carbonyls and amide groups. In the two-stage model of membrane protein folding, insertion of hydrophobic TM helices is followed by helix association (24, 25). As a result, helix-to-helix packing is a key element in defining the tertiary and quaternary structure of most membrane proteins and membrane protein complexes.

The folding of soluble proteins is thought to be driven by the hydrophobic effect and the increase in entropy associated with burying hydrophobic residues in the protein interior. Within the hydrophobic protein core, van der Waals interactions contribute significantly to the tight packing geometries that are associated with final folded protein structures. For TM helices of integral membrane proteins, the hydrophobic effect is lost as a driving force for helix association once the helices are inserted into hydrophobic bilayers. Helix association occurs through a combination of hydrogen-bonding, electrostatic, and van der Waals interactions. Unlike soluble proteins, membrane proteins rely on internal polar pockets for protein function; catalytic residues and ligand binding sites are often buried in the protein interior. This geometry then raises the question as to whether the interiors of membrane proteins are loosely packed or whether different amino acids are responsible for the packing of the helical segments of membrane and soluble proteins. The method of occluded surface (OS) (5, 8) (Fig. ​(Fig.1)1) was chosen to address these questions because it provides a direct measure of molecular packing and allows the fractionation of the atomic or molecular surface. Of importance is that the packing interactions can be quantified at the atomic, amino acid, or molecular level and both buried and surface exposed residues may be analyzed directly in contrast to the more commonly used Voronoi procedure (1). Moreover, the OS analysis promises to be a useful tool for probing the functional role of polar and conserved residues in the interior of membrane proteins by revealing how the neighboring and most closely associated residues (and atoms) pack.

Figure 1

Schematic of the OS calculation for the methyl group of threonine. The diagram depicts the normals extending from the molecular surface associated with the methyl group. The surface normals terminate if they encounter the van der Waals surface of residues within 2.8 Å.

^{The soluble proteins were classified according to Michie} et al. (26).

^{The average packing value for each protein class was determined by summing up the packing values of all amino acids in their helical sections and dividing the sum by the total number of amino acids in the helical section of each protein class.}

^{The soluble proteins were classified according to Michie} et al. (26).

^{See text.}

Acknowledgments

Abbreviations