BCHM 3010 Protein Structure Part II
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This 5 page Class Notes was uploaded by Morgan Dimery on Monday January 25, 2016. The Class Notes belongs to 3010 at a university taught by Dr. Cheryl Ingram-Smith in Spring 2016. Since its upload, it has received 17 views.
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Date Created: 01/25/16
Protein Structure Continued Intrinsically ordered proteins can either be fully disordered native or just have certain regions that are disordered Native intrinsically ordered proteins do not have a secondary structure at all Proteins that only have certain regions that are disordered just haven t gone all the way with their folding they look like a molten globule These proteins are very extended and have a lot of exibility because of this they are able to make contact with a lot of things very high surface area It is like they are giving a big hug They contain a lot of charged residues amp proline but do not contain a lot of hydrophobic residues because they don t have a way to hide them like normal proteins do Their ability to bind to target ligands very extensively has been thought to make them more stable Understanding how proteinfolding works is still a great area of study Denaturation is when the protein loses its 3D structure and function 0 The protein begins to unfold 0 Heat pH organic solvents detergents and urea or guanidine hydrochloride can cause denaturation of proteins 0 Cells go to great lengths to keep their proteins folded Renaturation is the refolding of a protein to bring it back to its normal conformation 0 Proteins can usually only recover from heat if they are exposed to it for a short amount of time At first the protein will start to unfold slowly and then as time goes on the protein will give upquot all at once denaturation is cooperative Ribonuclease A is a protein that chops upquot RNA it is hard to get rid of because it is very good at rebuilding itself Protein folding is not just done by trial and error Primary structure dictates protein folding The secondary structures formed will interact with each other to form intermediates As the intermediates become more and more complex the native final conformation is achieved Protein folding can be illustrated using a funnel Unfolded proteins are at the top of the funnel As you go further and further down the funnel the structures become more and more stable The native structure is at the bottom of the funnel and it is as stable as it can get The order of folding isn t necessarily set but there are more constraints as to what can fold hence why the funnel gets more narrow as you go further down the funnel How proteins fold can differ from protein to protein different types of funnels Sometimes the intermediate is so stable that the protein takes awhile to fold past it Difficulties can occur and the protein may aggregate instead of folding properly like a crumpled piece of paper it is completely useless MOLECULAR CHAPERONES AND CHAPERONINS Chaperones help proteins that have started to fold improperly If the protein hasn t gone too far down the wrong path then the chaperone can save it and steer it to the proper direction Chaperones can bind to the aggregated proteins to fix them Chaperones are usually found bound to hydrophobic areas that are exposed to the aqueous environment if hydrophobic regions are not hidden then there is a problem Chaperones block certain paths that are not good for the protein to go down Chaperonins do the folding for proteins that can t do it themselves Chaperonins put the protein into an isolation shell and give it a happy placequot to figure out what it wants to do and then release it to interact with other proteins Protein misfolding causes many human diseases The misfolding causes amyloids to form these cause insoluble aggregates of proteins 0 Examples Alzheimer s Parkinson s Huntington s mad cow amp dementia Usually the protein starts off as normal and then overtime it begins to misfold and cause problems Misfolding of another protein causes misfolding of another protein self perpetuating problem Protein Purification First you need to decide what protein you want to isolate you have to break the cell open to do this Most of the time there isn t going to be a lot of the protein present that you want they can be produced recominantly in other cells that are easier to grow such as E coli The main purpose of protein purification is to remove unwanted proteins without removing any of the protein you want to keep It is a trial and error process Centrifugation is a way to separate proteins by their size and density 0 It removes membranes unbroken cells and other junk o The broken cells are spun at a high speed and the heavy material goes to the bottom of the tube 0 A liquid supernatant will be present and this is where you will find the protein you want an additional filtering method can be used to further separate the protein pieces by their size Salting out is a method that separates proteins based on their differences in solubility o Ammonium sulfate is added in increasing amounts 0 Since proteins have different solubilities some start to come out into the tube very soon and others take longer di erent proteins precipitate from the solution at di erent salt concentrations 0 Once they come out they can be removed by centrifugation 0 This process makes the protein more pure adding liquid back causes it to go back to the impure way it was before Column chromatography is used to separate proteins by ion size hydrophobicity and a inity for ligand binding There is a stationary phase which is the part that contains beads There is a mobile phase which is the part that includes the liquid that ows over the proteins Proteins interact differently throughout the tube which causes them to move at different speeds o A lot of interaction in tube moves slow 0 Not much interaction in tube moves fast Different tubes are used to collect the different proteins Size exclusion chromatography gel filtration separates proteins by their size 0 This process uses porous beads beads that have pores o A liquid is added that causes the proteins to move around small proteins can enter the pores in the beads but large proteins cannot therefore they come out faster 0 Different tubes receive different volumes first fractions will have larger proteins Ion exchange chromatography separates proteins by their differences in charges of their R group 0 Charged beads that interact with proteins are used 0 Anion exchangers have positively charged beads that bind to proteins with a net negative charge 0 Cation exchangers have negatively charged beads that bind to proteins with a net positive charge 0 A salt solution is added that causes the proteins to be unbound again the more salt needed to get the protein off the more highly charged the protein was 0 Since proteins elute at different times they can separated based on charge strength Affinity chromatography separates proteins based on hydrophobicity or affinity for ligand binding 0 Hydrophobicity I Not all hydrophobic residues get hidden during protein folding I Hydrophobic beads are used I Proteins are put into a high salt concentration solution I When salt concentration is high there is a strong interaction between the beads and the hydrophobic regions of proteins that are still exposed I The salt concentration is then lowered and the proteins come off the beads I The proteins that have more hydrophobic regions will stay on the beads longer than those that have fewer hydrophobic regions 0 Affinity for ligand binding I Beads used have a ligand and only proteins that bind to this ligand will stick to these beads I Binding is so specific that this method can usually purify a protein in one step I An example of this method is called nickel a inity chromatography His binds to beads that have a nickel ligand they are removed by adding imidazole This method is used for purifying recombinant proteins they are produced with a long string of His so that they are easier to purify SDSPAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SDS reduces the effect of a charge on a protein by putting a negative charge onto the overall protein 0 Now that all of the charges on the proteins will be the same size is the only factor that in uences migration As the protein unfolds it is denatured with heat SDS and a reducing agent the reducing agent will remove the disulfide bonds in Cys so that the protein can completely unfold Mobility volts net charge size These proteins are now put on a acrylamide gel this gel is able to polymerize and cross link to form a gel with small pores The proteins try to weave through the pores and are separated by size Known molecular weights are used for comparison so the size of the protein can be estimated When the gel shows a single band you know that the protein is completely pure Protein size can be determined by making a graph I Xaxis distance protein migrated I Yaxis log of molecular weight of protein The molecular weight is determined by comparing it to a standard curve of known molecular weights A protein purification table shows how well each step of the protein purification process worked 0 There must be a good balance between losing your protein and enriching your protein The volume total amount of protein and activity will decrease after each step The specific activity will increase after each step if the steps are efficient You check for efficiency by comparing how much specific activity went up vs how much overall activity went down Overall the table will help you decide which steps worked well and which ones you shouldn t use in future purifications Enzymes speed up chemical reactions measuring the chemical reaction can tell you how much enzymatic activity there is O O 0 Total activity total units of activity in the solution Specific activity units of activitymg of protein Specific activity should increase as you purify a protein it doesn t matter that the total amount of protein and activity goes down
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