BIOL 1510 Chapter 11

Fluid mosaic model

a model of cell membranes that states that membranes are made up of a diverse array of molecules, including proteins and lipids, which can diffuse laterally throughout the membrane

Protein-lipid interactions

Proteins can recognize specific lipids within a membrane, and can recruit other proteins to that site

Importance of protein-lipid interactions

signaling and asymmetrical division

What happens when lipids are placed in an aqeuous solution?

They spontaneously form micelles, vesicles, and bilayers

Why do lipids spontaneously self assemble into structures?

It is thermodynamically favorable→even if micelles/vesicles are more ordered, the surrounding water gains disorder which is favorable

What types of lipids for micelles?

Conical lipids

What types of lipids for bilayers and vesicles?

Cylindrical lipids

• form bilayers first but then in order to hide the exposed sides they self assemble into vessicles

How can one study protein-lipid interactions in the lab?

Can create artificial membranes using DOPC, and incorporate proteins of interest into artificial membranes

Features of biological membranes

• sheet-like enclosed spaces

• made of lipids and proteins

• lipids are mostly amphipathic

• proteins mediate unique functions

• membranes are not freely permeable

• sheets self assemble, driven by hydrophobic interactions'

• lipids and proteins are asymmetrically divided across membrane surfaces

• membranes allow proteins to float in two dimensions

How do inner and outer membranes differ?

contain different lipids and proteins. Can differ in electrochemical composition/gradients

What lipids can induce curvature?

cardiolipin

What lipids can signal danger?

Phosphatidyl serine, if exposed to the outer membrane, signals danger

What organelle is enriched with cardiolypin?

Mitochondria→due to endosymbiotic origin

Peripheral proteins

Proteins found on one side of the plasma membrane

How do you isolate peripheral proteins from membranes?

Wash off with high salt or pH changes, which disrupts electrostatic interactions

Integral proteins

proteins embedded in membrane

How do you isolate integral proteins from membranes?

Must use detergents to solubilize

Lipid-linked proteins

Proteins tethered to the membrane through lipid linkages

How do you isolate lipid-linked proteins from membranes?

Use detergent or cleave off with enzyme

Types of lipid linkages

• GPI

• farnesyl

• palmitoyl

• miristoyl

Hydropathy plots

• plot amino acid residues on x axis

• plot hydropathy index on y axis

• look at the free energy change of putting an amino acid in an aqueous solution to determine hydrophobic vs hydrophilic parts of proteins

Are hydrophobic amino acids positive or negative on hydropathy plots?

They’re positive and above the x-axis→signify transmembrane residues!

Are hydrophilic amino acids positive or negative on hydropathy plots?

Negative and below the x axis→signify cytosolic or extracellular residues

What types of residues are found in the transmembrane portion of proteins?

hydrophobic

What types of residues are found in the cytosolic/extracellular portion of proteins?

charged residues

What types of residues are found at the interface of transmembrane and cytosolic portions of proteins?

hydrophilic but not charged→alcohol and amine amino acids

Common structure of pore-forming proteins

beta barrels

Why alpha-toxin is toxic

Form beta barrels in bacterial membranes, forming pores and allowing cell contents to leak out

What determine whether membranes or crystallized or fluid?

temperature and chain length, as well as presence of cholesterol

Are membranes usually highly ordered or disordered?

Usually disordered but can have ordered segments called lipid rafts

Can lipids diffuse laterally? Can the flip?

Lipids can easily diffuse laterally across the membrane

Flipping across the bilayers is very hard

Enzymes that help flip lipids across the bilayers

• flippases

• flopasses

• scramblases

Flippases

Moves phosphatidyl ethamine and phosphatidyl serine lipids from the outer leaflet to the cytosolic leaflet in an ATP dependent manner

Floppases

ABC transporters that move lipids from cytosolic to outer leaflet in ATP dependent manner

Scramblases

move lipids either to cytosolic OR extracellular leaflet but always in the direction of equilibrium. No ATP needed

Human and mouse experiment that proved protein diffusion within a membrane

1. Fuse GFP-labeled mouse and human embryonic cells

2. See that proteins are divided cleanly down the middle

3. After time, proteins are evenly interspersed across the plasma membrane

Fluorescence resonance after photobleaching (FRAP)

1. label lipids with GFP fluorescence

2. Zap a small section of membrane with laser to bleach some GFP-labeled lipids

3. Watch the recovery of fluorescence to see lipid diffusion

Can all proteins freely diffuse within the plasma membrane? Why or why not?

No, not all. Some proteins are anchored in one spot via cytoskeleton elements

Lipid rafts

Areas of highly ordered plasma membrane, often enriched in cholesterol, glycosphingolipids, scaffolding proteins, caveolin, and GPI-linked proteins

Do proteins in lipid rafts usually freely diffuse?

Usually no. Anchored in place

Types of solute transport

• simple diffusion

• facilitated diffusion

• primary active transport

• secondary active transport

• ion channels

• ionophore mediated ion transport

Simple diffusion

Molecules diffuse straight across the plasma membrane without a protein and without any energy input

How are transporters like enzymes?

• Transporters lower the Ea barrier for transport

• They only work for specific substrates

• Follow M/M kinetics

• Can be saturated

How exactly is Activation energy lowered?

Paid for by binding energy

Facilitated diffusion

Pores that allow molecules to flow down concentration gradient with no energy input

Aquaporins

Transport water into/out of cell via simple diffusion

Is facilitated diffusion exergonic or endergonic?

Exergonic

Important amino acid residues in aquaporins

• His

• Arg

• Asn

Function of His in aquaporins

Act as a size exclusion gate, restricting the size of the substrate ONLY to water

Function of Arg in aquaporins

Arg residues act in a repulsive fashion to orient water and break up H-bonding in order to prevent proton hopping

Function of Asn residues in aquaporins

reorient water dipoles

GLUT1 transporter

transports glucose down concentration gradient

Steps of glucose transport via GLUT1 transporter

1. binding of glucose to GLUT1 in stereospecific manner infuces a conformational change on the transport protein

2. Glucose is released into the cytosol

3. Transporter revers back to original conformation

Is glucose transporter via GLUT1 reversible?

In principle yes, but glucose is phosphorylated to G6P so it can’t exit

Primary active transport

Hydrolyze ATP to move molecules up concentration gradient

Secondary active transport

Utilize an electrochemical gradient to move a molecule up its concentration gradient while allowing another to fall down its concentration gradient

Types of cotransport/secondary active transport

• symporters

• antiporters

Calculating deltaG of transporting a neutral molecule

• C2 is concentration of molecules at a destination

• C1 is concentration of molecules at the original spot

Calculating deltaG of transporting a charged molecule

• C2 is concentration of molecules at a destination

• C1 is concentration of molecules at the original spot

• Z charge of ion

• Psi is the transmembrane potential

If deltaG is positive…

going against gradient

If deltaG is negative

going with gradient

if Psi is positive…

going against gradient

If Psi is negative…

going with gradient

Can transport be exergonic if its going against the concentration/charge gradient?

Yes!

Calculating deltaG total of primary active transport

Get deltaG_transport from deltaG=RTln(C2/C1)

Calculating deltaG total secondary active transport

• one should be exergonic, the other should be endergonic

• get deltaG from deltaG=RTln(C2/C1)

4 basic types of transporters

• P-type

• F-type

• V-type

• ABC

P-type transport

Uses ATP to transport molecule against a concentration gradient. They key is that the intermediate is a phosphorylated transporter protein

Inhibitor of P-type active tranport

Vanadate→mimicks the transition state

Example of P-type transporter

sodium potassium ATPase

F-type transport

Pump protons to create ATP

Example of F-type transporter

ATP synthase

V-type transporters

pump protons in an ATP dependent manner in order to acidify a compartment

• not phosphorylated as an intermediate

What organelle are v-type transporters important for?

Lysosomes

ABC transporters

Proteins with an intracellular ATP binding domain that are responsible for shuttling various molecules across the membrane

• change conformation to let molecules in/out

Example of ABC transporter and its function

MDR1→ comes up in chemoresistant cancer tumors and is used to pump chemotherapy drugs out. Can also show up on BBB to pump toxic compounds out of brain’s circulation

Putting transporters together in glucose transport from the intestinal lumen

1. Na+/K+ ATPase sets up sodium and potassium electrochemical gradient by pumping 3 sodium out and 2 potassium in

2. glucose/Na+ symporter drives glucose up its concentration gradient from the intestinal lumen to the intestinal epithelial cell

3. glucose exits the blood via GLUT2 uniporter down its concentration gradient

Ligand gated ion channel

small molecule binds to a receptor, inducing a conformational change which allows ions to flow down their concentration gradient

Voltage gated ion channel

A change in transmembrane electrical potential induces a conformational change, opening the channel and allowing ions to flow down concentration gradient

What limits facilitated diffusion?

The rate is limited by the maximum rate of diffusion

How can you measure the transport of charged molecules?

Patch-clamp technique

Would a liquid-ordered or liquid-disordered state be favored when the hydrocarbon chains of fatty acids are shortened?

liquid-disordered

Would a liquid-ordered or liquid-disordered state be favored when cholesterol and sphingolipid content is increased?

liquid ordered

Do organelle membranes often contain glycolipids?

No!

What anchors integral proteins in the membrane?

Hydrophobic interactions

If an inhibitor prevented the hydrolysis of the terminal phosphate off of sodium potassium ATPase, what would be the effect on transport?

Na would bind but would not be released

What kinds of proteins will addition of urea remove?

peripheral→breaks H-bonds that anchor them to the plasma membrane

What kinds of proteins will the addition of phospholipase C remove?

Anchored proteins