Membrane Protein Structure and Function

Membrane Protein Structure and Function

• Membrane protein structures are challenging to determine because most knowledge comes from indirect methods.
• X-ray crystallography is the standard technique for determining protein structures directly, but requires ordered crystalline arrays.
• Membrane proteins must be purified in detergent micelles, which are often heterogeneous in size, complicating crystallization.
• Compared to soluble proteins, membrane proteins are harder to crystallize.
• Advances in x-ray crystallography and new techniques like cryo-electron microscopy have improved the ability to determine membrane protein structures.
• A growing number of membrane protein structures are now available at high resolution.

Bacteriorhodopsin: A Case Study

• Bacteriorhodopsin is a small membrane protein abundant in the plasma membrane of Halobacterium salinarum.
• It acts as a membrane transport protein that pumps protons (H+) out of the cell.
• It contains a single chromophore called retinal, a light-absorbing nonprotein molecule, contributing to the protein's deep purple color.
• Retinal is covalently attached to one of the transmembrane α helices.
• Upon absorbing a photon of light, retinal changes shape, which induces a series of small structural changes in the surrounding α helices.
• This process results in the pumping of one proton from the retinal to the exterior of the organism.
• In sunlight, thousands of bacteriorhodopsin molecules actively pump H+ out of the cell, generating a concentration gradient of H+ across the plasma membrane.
• The cell utilizes this proton gradient to store energy and convert it into ATP.
• Bacteriorhodopsin is classified as a pump, a type of transmembrane protein that actively transports small organic molecules and inorganic ions across cell membranes.

Plasma Membrane Reinforcement: Cell Cortex

• The plasma membrane is extremely thin, requiring nearly 10,000 membranes stacked to match the thickness of standard paper.
• Membranes are typically strengthened by a protein framework.
• Proteins attach to the membrane through transmembrane proteins.
• Cell walls in plants, yeasts, and bacteria provide shape and mechanical properties.
• Cell walls are composed of a fibrous layer made up of proteins, sugars, and other macromolecules encasing the plasma membrane.
• Cell cortices in animal cells stabilize the plasma membrane through a meshwork of filamentous proteins.
• This network is attached to the underside of the membrane, providing structural support.

Red Blood Cell Cortex

• The cortex of human red blood cells has a simple, well-studied structure.
• Red blood cells are small and possess a distinctive flattened shape.
• Spectrin is a dimeric protein, long and thin (about 100 nm in length).
• Spectrin forms a lattice structure that supports the plasma membrane and maintains the biconcave shape of red blood cells.

Protein Confinement: Membrane Domains

• Cells have developed methods to restrict the movement of specific proteins, creating functionally specialized regions known as membrane domains.
• Plasma membrane proteins can be tethered to external structures, such as molecules in the extracellular matrix and adjacent cells.
• Proteins may also be anchored to relatively immobile structures within the cell, notably the cell cortex.

Barriers to Protein Movement: Epithelial Cell Example

• Cells can create barriers to confine specific membrane components to distinct domains.
• In gut epithelial cells, transport proteins are localized to specific regions:
  • Apical surface: Transport proteins for nutrient uptake are confined here (facing the gut contents).
  • Basal and lateral surfaces: Other transport proteins for solute export to tissues and bloodstream are restricted to these areas.
• Tight junctions between adjacent epithelial cells maintain the asymmetric distribution of membrane proteins.
• Tight junctions are formed by specialized junctional proteins that create a continuous belt around the cell at contact points with neighbors.
• This belt creates a seal between adjacent plasma membranes, preventing the diffusion of membrane proteins past the junction.

Cell Surface Carbohydrates: Overview

• Carbohydrates are covalently attached to some lipids and most proteins in the outer layer of the plasma membrane.

Fluorescence Recovery After Photobleaching (FRAP)

• FRAP is a method used to measure the mobility and diffusion of membrane components (lipids or proteins).
• Membrane proteins are labeled using fluorescent antibodies or covalent attachment of fluorescent proteins.
• A small patch of the labeled membrane is irradiated with an intense pulse of light from a focused laser beam, irreversibly “bleaching” the fluorescent marker in the targeted area.
• After bleaching, the fluorescence in the area is monitored using a fluorescence microscope, tracking the recovery of fluorescence by measuring the time it takes for neighboring, unbleached fluorescent proteins to diffuse into the bleached region.
• The rate of fluorescence recovery serves as a direct measure of the diffusion rate of protein molecules within the membrane.
• Experiments indicate that cell membranes exhibit a viscosity comparable to that of olive oil, suggesting a significant degree of fluidity.

Isolation and Reconstitution of Membrane Proteins

• Membrane proteins are extracted from cells and purified from the extracted mixture.
• Purified proteins are incorporated into artificial phospholipid vesicles, allowing the protein to maintain its proper structure and functionality.
• Membrane proteins generally diffuse more freely and rapidly in artificial lipid bilayers compared to natural cell membranes.
• Cell membranes are densely packed with various proteins and lipids, which can hinder protein movement.
• The complexity and variety of lipids in cell membranes contribute to reduced mobility for most proteins.
• Many membrane proteins in cells are anchored or tethered to the extracellular matrix or cell cortex.

Advantages of Studying Membrane Proteins in Artificial Lipid Bilayers

• Studies using artificial lipid bilayers have provided insights into how membrane proteins interact with lipids and their dynamics in a less complex environment.
• They have improved knowledge of how the structure and organization of cell membranes affect protein function.

Essential Concepts

• Membranes create barriers that confine specific molecules to distinct compartments within cells.
• The lipid bilayer, composed of a continuous double layer of lipid molecules, provides the fundamental structure and barrier function of all cell membranes.

Explore the complexities of membrane protein structures and the techniques used to determine their functions. This quiz covers the challenges faced in crystallization and features bacteriorhodopsin as a case study of membrane transport proteins. Gain insights into advances in methods like cryo-electron microscopy and x-ray crystallography.