Lecture 19

Front 1

Why is it important to look at protein structure and folding?

Back 1

Structural characterisation links the primary sequence to protein topology. The primary sequence does not always give enough information, but it is more valuable to know secondary, tertiary and quaternary structures that are critical for function.

Front 2

How have Crystallography and X-ray diffraction been useful to identify protein structures?

Back 2

We can make concentrated solutions so that crystals of protein form. We can then subject the crystal to X-Ray bombardment and the beams scatter in pattern that reflects atomic structure, this is a deflection scatter pattern. We use strategically placed radiation sensors to generate electron density maps.

Front 3

What do electron density maps provide?

Back 3

Electron density maps provide a skeleton upon which one can build a model. We can understand how electron density gives rise to deflection pattern. Structural chemists can build amino acids that fit into observed electron densities. They can build ball/stick models and get 3D understanding of how atoms come together in a particular protein.

Front 4

What are some benefits of proteins binding to other molecules?

Back 4

We can ameliorate drugs based on structural determinants that show how proteins can interact with other, smaller molecules. Ligands are binding entities that form ligand-protein interactions. A ligand can be a small molecule, nucelic acid, protein or steroid and can change protein function, usually as allosteric switches by binding given proteins, changing the conformation and modifying the function. By examining the disassociation constant we can describe tendency to form complex and examine the protein-ligand interaction.

Kd=[Protein A][Protein B]/[Protein A - Protein B]

and a smaller Kd = higher affinity.

Antibodies can bind to very small peptide sequences, an epitope on an antigenic target which is very strong! This is very important for the immune system. They have low Kd and so very strong affinities.

Front 5

What are enzymes?

Back 5

Enzymes are catalytic proteins that reduce reaction energy. This may be time, heat etc to enhance the reaction so it occurs more avidly. They have more forward efficiency by decreasing Ea. Enzymes bind substrates to form products through binding domains, active sites, that are specific and critical.

Front 6

How does the quantification of catalytic efficiency reflect specific properties of a protein?

Back 6

We look at speed, the formation of products, etc. We learn about substrate affinities by examining some of the kinetics that are typical of enzymatic catalysis. The substrate binds to the active site of enzyme to form enzyme-substrate complex. We put substrate in proximity to another substrate that may be critical for product formation. Once product is formed, it leaves the active site and the enzyme converts substrate to product. The enzyme is never consumed in the reaction. Increasing the substate will increase product formation to a certain extent, the maximal velocity. Vmax is relative to enzyme concentration due to availability of the active sites.

Km= the [s] at 1/2Vmax. This is Michael's constant and doesn't change.

Front 7

What is the significance of calcium and calmodulin?

Back 7

Proteins can often change conformation, used as triggers for events that make cell function. Calcium sensors are dependent on Calmodulin conformations. Calmodulin binds calcium and changes its conformation so it can act as a calcium sensitive trigger. This shows how small molecules can modify proteins.

Front 8

How can the modification of proteins switch them on and off?

Back 8

Proteins that bind to guanine are great switches. They play critical role as triggers in the cell, GTP binding proteins. There is often also weak GTPase proteins and weak hydrolysis activity. Binding to GTP makes proteins take an active conformation, this can trigger specific processes linked to them, controlled by auxiliary proteins that shut down the GTP bound state. GTPase activating enzyme (GAPs) that can enhance GTP hydrolysing ability to GDP, putting it into GDP bound state which is generally off. This is negative regulation. We need guanine exchange proteins to get GDP out, replace with GT in the active conformation. This is positive regulation by GEFs. Proteins can also be phosphorylated. This changes charge and alters protein conformation. Protein kinase transfers phosphate to acceptor and gives a new function. Protein phosphates remove these modifications.

Front 9

What is centrifugation?

Back 9

It is a crude way of separating and purifying proteins. 2 types:

Differential: A mechanism to separate organelles and is preparative. You have an extract, put it in a test tube, centrifuge it at high velocities, large particles drop to the bottom, remove large/different parts by sequential steps. Keep removing the supernatant until we get purified nuclei. This depends on the velocity.

Rate-Zonal: Separation based on density. Uses sucrose. Expose to very high speeds for amount of time. Sedimentation at different levels that correspond to density shapes. Then test the test tubes for the different components.

Front 10

What is SDS-PAGE?

Back 10

Separates molecules by size by acting like a sieve. Take the complex protein sample, add SDS detergent, boil sample to denature the proteins and bind SDS. Put in the reducing agents. SDS is highly negatively charged, now so are proteins. We can run through polyacrymilide gel to separate the complex mixture by size through electrophoretic field. Then, stop voltage and remove gel, can then colour the gel with dye and then destain to show only the stained polypeptides. Loading size marker helps us immediately detect the peptide size.

Front 11

What is immuno-blotting or "Western Blots"?

Back 11

It can be hard to visualize bands but if you have a protein target and antibody we can use antibody to detect the protein of interest after transferring the protein sample to a solid matrix which we can hybridize with an antibody solution. Antibodies are important for cellular defences but are also critical proteins that recognize and bind to ligands with very strong affinities for given targets. To use antibodies we run protein samples using SDS page, transfer proteins that have been separated onto nitrocellulose filter, then incubate with antibody, remove non-specific antibody. Use 2nd antibody that recognizes the first antibody and uses enzymatic activity to show where 2nd antibody is enriched. We can then identify our protein of interest.

Front 12

What is 2D Gel electrophoresis?

Back 12

It is even more powerful and we don't get a giant smear. It separates protein mixture by charge. Use isoelectric focusing to separate then induce with SDS and run proteins in 2nd dimension to separate by mass. Put in pH gradient which uses strip of gel that contains polyanionic or polycationic molecules called ampholytes. It separates ampholytes that gives rise of low pH to high pH within gel. Can layer protein sample pH gradient and allow proteins to migrate to a point in pH gradient where they're neutral. This is isoelectric focusing. We can then place strip on SDS page gel, infuse SDS into strip and migrate proteins. Basically get x2 classification into polyacrymilide gel and organize into size.

Front 13

How does Gel filtration or Size exclusion chromatography separate proteins according to size?

Back 13

Use when you have a large sample and need to do preparative work. Use liquid or column chromatography to separate proteins by size with gel filtration or size exclusion. The way proteins interact with the column matrix shows size. The matrix contains pores/indentations that can be size regulated. Diffusion through column, small proteins will go into pores and take longer so that giant proteins come out first. Messing with the pore size allows for selection of which proteins can interact. Ellution is the process of running something through the column, collecting into small volume recipients, can combine to do multiple separations all using different properties of the polypeptides.