Unraveling a mystery: Why human cells require cholesterol.

Understanding how cholesterol binds to mammalian cells offers critical insights into the waxy substance's role in protein modulation and cell function.

Non-covalent binding of membrane lipids to membrane proteins.

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function.

Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated.

Membrane protein fragments reveal both secondary and tertiary structure of membrane proteins.

Structural data on membrane proteins, while crucial to understanding cellular function, are scarce due to difficulties in applying to membrane proteins the common techniques of structural biology. Fragments of membrane proteins have been shown to reflect, in many cases, the secondary structure of the parent protein with fidelity and are more amenable to study. This chapter provides many examples of how the study of membrane protein fragments has provided new insight into the structure of the parent membrane protein.

A small subset of signal peptidase residues are perturbed by signal peptide binding.

Perturbations of the chemical shifts of a small subset of residues in the catalytically active domain of Escherichia coli signal peptidase I (SPase I) upon binding signal peptide suggest the contact surface on the enzyme for the substrate. SPase I, an integral membrane protein, is vital to preprotein transport in prokaryotic and eukaryotic secretory systems; it binds and proteolyses the N-terminal signal peptide of the preprotein, permitting folding and localization of the mature protein. Employing isotopically labeled C-terminal E.

Solution NMR of signal peptidase, a membrane protein.

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export.

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch.

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface.

G-protein coupled receptor structure.

Because of their central role in regulation of cellular function, structure/function relationships for G-protein coupled receptors (GPCR) are of vital importance, yet only recently have sufficient data been obtained to begin mapping those relationships. GPCRs regulate a wide range of cellular processes, including the senses of taste, smell, and vision, and control a myriad of intracellular signaling systems in response to external stimuli. Many diseases are linked to GPCRs.

Asymmetric stability among the transmembrane helices of lactose permease.

Combining structure determinations from nuclear magnetic resonance (NMR) data and molecular dynamics simulations (MD) under the same environmental conditions revealed a startling asymmetry in the intrinsic conformational stability of secondary structure in the transmembrane domain of lactose permease (LacY). Eleven fragments, corresponding to transmembrane segments (TMs) of LacY, were synthesized, and their secondary structure in solution was determined by NMR. Eight of the TMs contained significant regions of helical structure.

Stability of loops in the structure of lactose permease.

Structural analysis of peptide fragments has provided useful information on the secondary structure of integral membrane proteins built from a helical bundle (up to seven transmembrane segments). Comparison of those results to recent X-ray crystallographic results showed agreement between the structures of the fragments and the structures of the intact proteins. Lactose permease of Escherichia coli (lac Y) offers an opportunity to test that hypothesis on a substantially larger integral membrane protein.

A conformational trigger for activation of a G protein by a G protein-coupled receptor.

G protein-coupled receptors (GPCRs) are a family of seven transmembrane helical proteins that initiate a cellular response to an environmental signal. Once activated by an extracellular signal, GPCRs trigger the intracellular signal transduction cascade by activating a heterotrimeric G protein. The interaction between the G protein and the receptor, which triggers the signal transduction, is the focus of intense interest.

Structural studies on rhodopsin.

Bovine rhodopsin is the prototypical G protein coupled receptor (GPCR). It was the first GPCR to be obtained in quantity and studied in detail. It is also the first GPCR for which detailed three dimensional structural information has been obtained.

Structural studies of metarhodopsin II, the activated form of the G-protein coupled receptor, rhodopsin.

The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin.