# Structural Biochemistry/Lipids/Biological Membranes
## Determining Membrane structure

Using a technique called freeze fracture electron microscopy scientist were able to discredit the membrane sandwich model by the discovery of transmembrane proteins. In this technique a membrane is frozen in liquid nitrogen and then shattered so the membrane splits between the leaflets of its bilayer exposing the interior of the lipid bilayer and its embedded proteins. Electron micrographs are then used revealing that the interior of the membrane is studded with globular membrane proteins and the outer surface has a relatively smooth appearance.

## Bacteria Cell Walls

Bacteria cell walls are composed of Peptidoglycan, which create rigidity of the cell wall, determine cell shape, and help prevent osmotic lysis. The bacteria cell wall, like most lipid bilayers, is porous and semipermeable. Gram positive bacteria have a very thick peptidoglycan layer and therefore, when dyed during gram staining, retain the crystal violet dye. Gram negative bacteria have a thin peptidoglycan layer which results in an inability to retain the crystal violet dye during gram staining.
Peptidoglycan, also known as murein, is composed of alternating units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). NAM is essentially a molecule of NAG onto which a lactyl group has been added to C3 via phosphoenol pyruvate; it is a signature molecule of bacteria. A pentapeptide chain is attached to the lactyl group during the NAM-NAG unit formation inside the bacteria. Generally, this pentapeptide chain consists of, in order: L-ala-D-Glu-mDAP/L-Lys-D-ala-D-ala. Once the NAG-NAM complex complex crosses the bacteria cell membrane into the periplasmic space, the terminal D-ala is cleaved off and the penultimate D-ala is cross linked to a DAP (forming a direct crosslink), or to a L-Lys (via a peptide interbridge) of another NAG-NAM complex. This crosslinkage is catalyzed by transpeptidases.
There are two cellular compartments that are involved in the biosynthesis of peptidoglycan. The formation, association, and assembly of soluble precursors onto a lipid carrier occurs in the cystoplasm. This generates Lipid II. This is a complex molecule that is thought to be translocated to the outer side of the cytoplamsimic membrane by integral membrane proteins of the shape, elongation, division, and sporulation family. The Lipid II's glycan chains are polymerized and the its stem peptides are cross linked. This is catalyzed by peptidoglycan syntheses which are penicillin-binding proteins, making this a target of B-lactam antibiotics. The biosynthesis of peptidoglycan involves over 10000 common reactions catalyzed in the cytoplasmic compartment.
The antibiotic penicillin works by competitively inhibiting the transpeptidase via the beta-lactam ring in the drug. Vancomycin also prevents the peptidoglycan layer from forming, but it works by binding to the terminal D-ala-D-ala complex, preventing transpeptidases from working. It typically is used as a last resort. Other drugs that work to inhibit peptidoglycan synthesis include phosphomycin, which inhibits NAM formation by competing with phosphoenol pyruvate, D-cycloserine, which inhibits enzymes that form the D-ala-D-ala unit inside the bacteria; and bacitracin, a drug that prevents bactoprenol from being recycled (bactoprenol is a carrier lipid, a homolog of dolichol, that moves the NAM-NAG complex across the cell membrane).
Additionally, bacteria have their own control mechanisms which inhibit peptidoglycan synthesis so that the organism can grow. These controls are classified as murein hydrolases, or autolysins, because they cleave various structures important for the murein layer. Hexosamidases cleave the beta-1,4 link between NAM and NAG, amidases cleave the peptide bond between the lactyl group and the pentapeptide chain on NAM, endopeptidases cleave various parts of the pentapeptide chain, and carboxylpeptidases cleave the D-ala carboxyl group, preventing crosslinking.

## Penicillin-Based Proteins

Once Lipid II is on the periplasmic side of the membrane, it undergoes the polymerization of the glycan chains and the cross-linking of the stem peptides. While the Penicillin-binding proteins with high molecular mass carry out both of these reactions, penicillin-binding proteins with low molecular mass can exert carboxypeptidase or endopeptidase activities. These activities cleave peptide bonds within the stem peptide in order to regulate the level of peptidoglycan cross-linking. This insertion of the newly synthesized chains of peptidogylcan into the pre-existing layer is accompanied by the turnover of the old material produced by hydrolases (transglycosylases and endopeptidases). This sequence has proposed the idea that penicillin-based proteins are themselves associated with cell wall degrading enzymes within a comples that is located at the interface between the membrane and the periplasm.

## Common Features that Biological Membranes Share
### Composition of Membranes

The fluid mosaic model says that the three main components that make up membrane bilayers are lipids, proteins, and carbohydrates. In general, most membranes are composed of approximately 75% lipids, 20% proteins, and 5% carbohydrates.

### Sheet-Like

Membranes are only two molecules thick and are sheetlike structures. They form closed boundaries and create different compartments. The thickness of most membranes are only between 6 nm and 10 nm.

### Built of Lipids and Proteins

The mass ratio of lipids to proteins can range anywhere from 1∶4 to 4∶1. Although most membranes are made mostly of lipids and proteins some can have carbohydrates that are linked to lipids and proteins.

### Composed of both hydrophilic and hydrophobic moieties (Amphipatic)

Membrane lipids are small molecules and they have both hydrophilic and hydrophobic moieties. The membrane lipids form closed bimolecular sheets. These lipid bilayers that are formed provide barrier to the flow of polar molecules. The hydrophilic head of the lipid molecule is on the outside surface of the cell membrane in addition to the inside surface of the membrane while the hydrophobic fatty acid long chains are on the inside of the lipid bilayer.

### Proteins mediate functions

Different proteins mediate different function for the membrane. The membrane proteins are embedded in the bilayer and serve as pumps, channels, receptors, energy transducers, and enzymes.

### Noncovalent

The proteins and lipids in the membrane are held together through noncovalent forces that work cooperatively.

### Fluid

Membranes are fluid and lipid and protein molecules can freely diffuse into the plane of the membrane but do not rotate across the membrane. The membranes are two dimensional planes of oriented proteins and lipids.
Membranes are not static sheets of molecules locked in place but rather, they are held together by hydrophobic interactions which are much weaker than covalent bonds. As a result, most of the lipids and some of the proteins can diffuse laterally across the membrane. Adjacent phospholipids switch positions about 10$^7$ times per second where as molecules flip-flopping transversely across the member is rarely observed. The reason flip-flopping is rarely observed is because switching from one phospholipid bilayer to another requires the hydrophilic part of the molecule crossing the hydrophobic core of the membrane. Proteins on the other hand do drift laterally but at a much slower pace as their size, compared to lipids, is relatively large. Two factors increase the fluidity of membranes. The first factor is the presence of unsaturated hydrocarbon tails of phospholipids which have kinks that keep the molecules from packing closely together. The second factor is the presence of cholesterol which enhances fluidity at low temperatures by hindering solidification by disrupting the regular packing of phospholipids. What also enhances the fluidity of lipids is the lateral (fast) and flip flop (slow) exchange of place of lipid molecules between each other.

### Asymmetric

The membrane does not match up and is asymmetric.
Asymmetry of membranes result for different functions needed by the cell as well as the molecular make up of each membrane. A membrane is a collage of different proteins embedded in the fluid matrix of the lipid bilayer. Phospholipids make up the majority of the membrane but proteins determine most of the membranes asymmetric properties as a result specification of membrane functionality. Different types of cells contain different sets of membrane proteins and the various membranes within each cell have a unique set of proteins necessary to carry out specific functions. An example of this is the sodium and potassium ion pump which uses ATP as a source of energy to pump ions in and out, but in order to do so, ATP must be inside of the cell to drive it.
There are two major populations of membrane proteins, integral proteins, which penetrate the hydrophobic core of the lipid bilayer, and peripheral proteins, which are no embedded in the lipid bilayer at all. Certain membranes contain distinct orientations of their integral and peripheral proteins, adding to the asymmetric character of the membranes. They have unique orientations because they are inserted into the membrane in an asymmetric manner after synthesis. Many of the integral proteins are trans membrane proteins, which completely span the membrane. The hydrophobic regions of an integral protein consist of one or more stretches of nonpolar amino acids usually coiled in alpha helices. The hydrophilic parts are exposed to the aqueous solutions on either side of the membrane. Peripheral proteins are loosely bound appendages on the surface of the membrane and are usually exposed to parts of integral proteins. Depending on the functions needed by the cell, whether it be transport, enzymatic activity, signal transduction, cell-cell recognition, intercellular, attach to the cytoskeleton and extracellular matrix, each membrane will look different. This asymmetry is preserved because membrane proteins do not rotate from one side to the other but "are always synthesized by the growth of preexisting membranes" (Berg, 345).
An example of membranes differing in their protein content is Myelin. Myelin is a membrane that serves as an electrical insulator around certain nerve fibers and has a low content of protein, 18%. Compared to the protein content of plasma membranes, 50%, the protein content is relatively low in Myelin. There are three major kinds of membrane lipids phospholipids, glycolipids, and cholesterol. The content of each in a membrane depends on the functions needed by the membrane and the characteristics of the cell.

### Electrically Polarized

The inside of the membrane is negative at around −60mV.

## Cell Growth and Forces that Shape It

Cell wall elongation and division of multi-protein complexes usually involves large macromolecues that are found on the outside of the cytoplasmic membrane. Penicillin-binding proteins, (PBPs) are intimately associated to the cytoplasmic cytoskeleton proteins. The bacterial cytoskeleton is a very important aspect of bacteria, and is the target of penicillin finghting antibiotics. Peptidoglycan is the macromolecule that determines the shape and maintenance of a bacteria cell by influencing the growth of the bacteria cell wall. There are two biochemical reactions that occur in the terminal stage of the peptidoglycan synthesis that is responsible for the lateral wall elongation and for septation. The two peptidoglycan-synthesizing systems are in competition with each other so that there is not any peptidoglycan synthesis for lateral wall elongation occurring during septation and not septation during cell wall elongation. The final shape of the bacteria is determined by these two cell wall synthesis interactions. A normal balance between the two reactions with result in bacteria being shaped like rods. An unusual prevalence of the site for elongation will lead to long rods or filaments. Any prevalence of septum formation will lead bacteria to the formation of coccobacilli or cocci. However, bacteria has a negative control that blocks septum formation when lateral wall elongation is not completed. This will help keep cocci from formation. Mecillinam is a Beta-lactam antibiotic that will specifically bind to penicillin, binding protein 2 (PBP 2). This will inhibit lateral wall elongation and transform rod shaped bacteria into cocci shaped bacteria. This can be extremely useful as the cocci shaped bacteria is unable to proceed to further division. However, it has been found that re-adding mecillinam to cocci that have not yet reshaped into rod shaped bacteria inhibits the peptidoglycan synthesis but does not interfere with the division occurring in the cells to which the antibiotic has been re-added even though these cells divide as cocci. This indicates that in order to release septum formation form the control that the lateral cell wall exerts on it, the cell wall does not need full elongation of the lateral wall. It has been found that cells with the removal of mecillinam, roughly one hour after removal, the cell division occurs. This suggests that the time needed for the septum to start and fully complete is approximately an hour. Because a normal rod will divide in half the time it takes for septum to complete, it is possible to deduce that the two septa are initiated at the same time in exponential cells growing under these conditions and are then stopped by the lateral wall elongation beginning. However, septum formation can be blocked by intracellular event such as DNA replication that postpone septum termination until such time as they are complete.

## References

* Berg, Jeremy M. 2007. Biochemistry. Sixth Ed. New York: W.H. Freeman. 327
* Campbell, Neil A., Reece, Jane B. 2005 Biology Seventh Ed. Pearson Education, Inc.