Lack of Hydrophobic Drive in 7TMRs

Water-soluble globular proteins adopt reasonably restrained formal structures during the folding process because of hydrophobic drive.9 The generally hydrophobic nature of zwitterionic amino acids is worsened by the elimination of water during peptide bond formation in the production of extended chains of residues. Hydrophobic collapse ensures that the more hydrophilic residues are surface-exposed and many secondary modifications are exploited to increase aqueous solubility, including gly-cosylation and phosphorylation. Secondary and tertiary structures are constrained by this hydrophobic challenge, producing autonomous domains of a limited size range and resulting in the marked proclivity of shorter peptide sequences to copre-

cipitate or infiltrate membranes. The low dielectric constants of a lipid bilayer and of a protein interior, e = 2 to 5, are indistinguishable in comparison with the high value of e = 80 for water. This inability to discriminate between the interior physical properties of a protein and of a plasma membrane means that the orientation of transmembrane peptides cannot be derived reliably using predictive programs.10 Thus, the membrane-resident portions of proteins are relieved of an aqueous imposition on their conformation.

The power of the hydrophobic drive is ineffectual in the lipophilic environment of the cell membrane and there is much less constraint on protein internal movement, resulting in an inevitable enhancement of flexibility. Nevertheless, the spate of prokary-otic membrane protein structure determinations in recent years ( was made possible by selective "tricks" that restrict protein flexibility. These include coprecipitation with antibody Fabs, site-directed mutagenesis (SDM), sequence truncation, chemical modifications, and "ligand rescue." For example, the 12-helix protein lactose permease, LacY, is a favored model for the multidrug transporters (MDRs or P-glycoproteins) responsible for small molecule metabolism and drug expulsion by cells. The broad specificity of MDRs for a wide range of molecules already implies some flexibility of recognition and response. Models of the permeases and MDRs were provided by distance constraints from many concerted approaches (e.g., a PubMed search lists 50 P-glycoprotein publications for Loo, T.W. and Clarke, D.M. in the past decade), including exhaustive tandem SCAM (scanning cysteine-accessibility mutagenesis) to generate a multitude of dithiol bridges. The ultimate elucidation of the crystal structure of LacY (1pv7) was only possible after the rational removal of a specific cysteine residue (C154G), to prevent nonspecific aggregation, and by the inclusion of substrate, which acted as a folding nucleus and preserved the structure during purification by ligand rescue. The water-filled channel is occupied by lactose and the binding site is provided by helices 1, 2 and 6, provided by both of the two autonomous bundles of six distorted helices.

The crystal form of rhodopsin (Figure 10.3B) loses its integrity on exposure to red light, as the chromophore dissociates after light activation and the crystals lose their diffractive ability.11 Thus, even the reasonably rigid rhodopsin structure becomes more fluid on ligand loss, suggesting that flexibility is an intrinsic feature of 7TMRs. Given the low rates of successful crystallization for multispanning membrane proteins and the consequent paucity of structural information, it is all the more surprising that unrelated heptahelical membrane protein structures have been largely ignored by the 7TMR community.

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