VCU CHEM 403: Biochemistry Exam 2 Notes- Roesser PDF

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Professor: Roesser...

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Lecture 1 - 2/3 - Peptide Basic peptide structure (it’s supposed to be terminus but I’m too lazy to change it)

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Charges of peptides relies solely on the R groups - net charges are determined by the side chains side groups ● Ex: If you have a lot of lysine or arginine the peptide is going to be positively charged at neutral pH ● Ex: If you have a lot of glutamic acid or aspartic acid the peptide is going to be negatively charged at neutral pH - Everything about the peptide depend on the sequence of side chains 1) Give the peptide it’s structure 2) Gives the peptide it’s function - If a polypeptide has a function it’s going to be a protein Nomenclature of peptides - 2 amino acids = dipeptide - 4 amino acids = tetrapeptide - 5 amino acids = pentapeptide - 2-20 amino acids chains are considered oligopeptides - Greater than (>) 20 amino acids chains are considered polypeptide Example: Draw the following peptide ala phe tyr val

Resonance structures of peptide bonds

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In the resonance structures theresa double bond, which means peptide bonds have partial characteristics of double bonds - Characteristics of a double bond ● More energy of stabilization (takes more energy to break the bond) ● Shorter bond length ● No free rotation around the bond ○ This one is particularly important because when you look at the general structure you can see there is a limited amount of conformations because of double bonds Possible number of amino sequences in a protein = 20N - Where N is the number of amino acids in the protein - This shows us that there's lots and lots of sequences, and that the number goes up exponentially - Ex: 100 amino acids are in a protein: 20100 is the number of sequences 1

What are the sizes of proteins inside of the cell? - 10 kilodaltons (KD) to 100 KD ● “Ten thousand molecular weight - a hundred thousand molecular weight” - Rosser - Minimum size requirement (minimum limit) ● Textbook: Need a large enough peptide/polypeptide to fold into a specific, active structure ● Rosser: Cellular proteins must be regulated ○ The regulation contributes to size - Maximum size requirement (Maximum limit) ● Textbook: This is as large as a ribosome can make it without making too many errors ● Rosser: Time - how long does it take to make a large protein ○ If you look at 100 KD protein, it's about 17 amino acids per second for the average ribosome. That means it takes about 30 minutes a 100 KD protein ○ Bacteria can divide every 30 minutes, so why would they want to make a protein that takes a generation to make? The answer is they don’t because it doesn’t make sense for them to do so ○ The point of this is to say that cells don’t want to spend long amounts of time creating proteins “Handy to know for the course” - The average molecular weight of an amino acid is 110 Daltons ● That means if you have 100 amino acid proteins (100 x 0.11) there would be about 11 KD ● If you had 40 KD on a gel you could guesstimate how many there is - It’s about 440 amino acids probably (40/0.11 = 363.6 amino acids) Protein structures - Proteins require specific overall structures for activity ● This overall structure is the lowest conformation of the protein - Levels of structures: 1) Primary structure (1°) - sequence of amino acids in the peptide chain - Held together by peptide bonds (aka covalent bonds, aka strong forces) - By just having amino acids linked together by peptide bonds does not guarantee activity 2) Secondary structure (2°) - form between amino acids that are close together in the primary sequence - Held together by hydrogen bonding (a weak force) - Two types: ● 𝛼 helixs ● 𝛼 sheets 3) Tertiary structure (3°) - overall structure of polypeptide - Now you can get interactions between amino acids that are not close together (long range interaction) - Held together by all of the weak forces, but primarily by hydrophobic interactions - Putting a nonpolar residue away from the polar water, and putting the ionic polar amino acids on the outside to interact with water ● Doing it this way makes it soluble ● If you disrupt (by boiling) the higher level structures, they don't fold properly, and their nonpolar amino acids are on the outside. This causes them to fall out of solution and they are no longer soluble Lecture 2 - 2/12 - Polypeptides Peptide bond - Is a partial double bond - meaning there is no free rotation around it Protein classes 1) Fibrous proteins - they have a simple linear structure - Most are not soluble in water - Tend to be structural protein - they give structural integrity (don’t let things float away) - Ex: Actin - can be polymerized into a fragment - they give structure to the cell - Never going to talk about fibrous proteins after this, so don't focus too hard on this section 2

2) Globular proteins - they are spherical/ semi-spherical in shape - Water soluble in water ● They form micelles, which makes it water soluble ● “Nonpolar residues inside; polar and charged amino acid outside - Do the chemical work in the cells ● They are the enzymes ● When we talk about kinetics it’s going to be almost all globular proteins 3) Membrane associated proteins - Not water soluble because membranes are very nonpolar ● This causes MAP to be nonpolar which causes them to be not water soluble - Function: transporting molecules (they help transport things from one side to another), receptors (things like hormones in your bloodstream react with receptors which tend to be membrane bound proteins) Layers of protein structured - aka globular structures 1) 1° - sequence of amino acids held together by peptide bonds (strong force 2) 2° - Form between sequences that are very close together - Held together by Hydrogen bonds - helices and 𝛼 sheets 3) 3° - Overall structure - Long range interactions - Held together by all weak forces, but primarily hydrophobic interactions ● Hydrophobic interaction make low energy situation, also makes the protein soluble - If you disrupt a 3° structure the protein will not be active, many times will participate out of solution, and will not be soluble 4) 4° - Majority of cells in our body have more than one polypeptide/subunit - Held together by all weak forces Exception to higher order being held together by only weak bonds - Minority of 3° and/or 4° may be held together by a strong force via cysteine-cysteine disulfide bonds (a covalent bond)

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Most proteins that have disulfide linkages are secreted outside of the cell ● Ex: Bacteria secrete proteins out into the soil or water so they’ll release proteins with disulfide bonds. These bonds are stronger and can withstand a harsher environment ● Ex: Antibodies - they have 2 light chains and 2 heavy chains. Subunit and individual peptides are held together by disulfide bonds. a) Antibodies get released into the bloodstream and can float out there for months, so it’s important that they are stable Multisubunit proteins - In a vast majority of cases there is an even number of subunits Nomenclature of multisubunit proteins - Ex: Trp receptor � � ● Called a homodimer ● Homo because same subunit, dimer because there's two of them 3

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Ex: Hemoglobin 𝛼 𝛼 𝛼 𝛼 �

𝛼 𝛼

● Called heterotetramer Advantages of being a multisubunit 1) Allows for larger proteins - There's a size range for polypeptides (~100 KD), but if you have 8 subunits at the same molecular weight (~100KD) then you have a much bigger protein (8 x 100 = 800 KD) - Multisubunit takes less time to make the more cells 2) Genetic economy - not applicable for humans - “having compact DNA is important” - Roesser - Takes 30 minutes for E.coli to replicate so 99% of its DNA codes for protein. There's very little wasted DNA - This is because they are constantly competing with others and can win by growing the fastest - Ex: If you have a homotetramer vs a single polypeptide

� � 𝛼 𝛼

𝛼 𝛼 𝛼 𝛼

𝛼𝛼 𝛼 𝛼 𝛼𝛼 𝛼𝛼

● For homotetramer - each polypeptide subunit (4) is 150 amino acids. There are 3 nucleotides per amino acid. Takes about 450 nucleotides in DNA to encode for the protein ● For large single polypeptide - It's the same size as the subunit, has a total of 600 amino acids so it takes 1,800 nucleotides in DNA to encode for the protein ● This shows that less DNA is needed for subunits. This is important for E.coli and stuff because it takes less effort 3) Efficiency - think of an assembly line - Ex: Trp synthase in E.coli molecule ● Tryptophan is the hardest amino acid to make. The last 2 steps are as follows:

● Trp synthase does all of this in subunits. It passes it on from one to the next ○ This is huge because the cell doesn’t need 2 enzymes to create the amino acid and it doesn't waste time releasing one part into the cytoplasm and waiting for it to find its counter.

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Key: Trp Synthase 𝛼 𝛼 Indole Glycerophosp hate

Ind

𝛼 𝛼 ole 𝛼 𝛼

𝛼 𝛼

Tryptop

4) Cooperativity han - Form on enzyme regulation that requires multiple subunits - This is a very precise way to regulate subunits Lecture 2 - 2/14 - Purifying proteins Overview of multisubunit advantages 1) Larger proteins: exactly what you think 2) Genetic economy: Having compact DNA - which is important for prokaryotes and microorganism but not for us 3) Efficiency: how effective is the cell is at passing on from one step to the next 4) Cooperativity: form of enzyme regulation that requires enzyme to have more than one subunit - When something binds to enzyme or protein a conformational change in the protein occurs this is called a ligand - Ligand: ● Can be a substrate ● Can be a molecule that is called an effector that regulates activity ● It changes the conformation of the enzyme ● Ex of cooperativity: Hemoglobin 𝛼

𝛼 𝛼

𝛼

○ Heterotetramer (the and are related) ○ Each of the subunits have an heme-Fe (heme-iron) at the active site ■ This heme-Fe binds to O2: a fully saturated heme-Fe binds 4 O2 ■ Once O2 binds to a subunit it causes a conformational change in the rest of the subunits ● This causes remaining altered subunits to have lower affinity for binding O2 (this is cooperativity) ■ Hemoglobin has to do two things 1) When blood is going through your lungs and there is a high oxygen concentration, you want all 4 of heme-Fe sites to bind an oxygen (high oxygen = high affinity to bind) 2) When blood is flowing to other tissues, which have a much lower oxygen concentration, you want as much oxygen to be released by hemoglobin to go to the tissues (low oxygen - low affinity to bind) Purifying proteins: used to figure out how protein works, what its mechanism is, how much its regulated, etc - “Don't waste your clean thinking on dirty proteins” - isolate proteins then study them 5

What do we need in order to purify proteins? 1) You need a source of protein from various cells - Lyse the cells and make a protein extract from the cell 2) Need a specific assay for “our protein” (we’re calling it “ours” to keep it vague) 3) Need to know where the protein is and is not - if you don’t know this you can’t purify 4) Need an indicator - this tells whether there is that protein in the sample or not and that is very important - The simpler the better Example of an indicator: �-galactosidase - Enzyme that takes lactose (disaccharide) and cleaves it into glucose + galactose - Long time ago someone synthesized x-gal, which is a synthetic substrate (synthetic lactose basically) ● 𝛼-galactosidase can cleave x-gal (clear in color) into glucose + x-galactose (deeply blue in color) ● If you do a purification and you want to know where -galactosidase is in 100 fractions, you take a little bit of each fraction and mix it with x-gal ○ If it turned blue then you have �-galactosidase ○ If it stays clear then there is none Steps to purifying a protein 1) Take protein extract and separate out by some physical method (physical characteristic) to create fractions 2) Assay all the fraction - To see where certain proteins are and where it’s not 3) Combine the fractions that have the protein and move onto next separation step 4) Keep repeating these steps until you get a pure protein Steps for classical biochemical purification - Classical biochemistry is most often used for protein complexes (purifying them) 1) Separate by solubility: Take protein extract and add a salt to it, usually ammonium sulfate (NH4)2SO4 - Reminder: that soluble proteins are soluble because their polar and charged residues on the outside and their nonpolar uncharged residues on the inside

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+ ++ + -+ + +- +-

+-

If you add salt to it, ● NH4 is going to associate with (+) charge on protein ● SO4 is going to associate with (-) charge on protein ● Once most or all charges on the outside of the protein on the surface are neutralized it will precipitate and fall out of solution - Precipitation will occur at different concentration of ammonium sulfate for different proteins ● Ex: Solution is 20% saturate in ammonium sulfate, you add the needed amount of ammonium sulfate to protein extract ○ Some proteins will start to precipitate out some will still be soluble ○ Spin it and separate the solids from the soluble proteins ○ Put soluble proteins back in solution dissolve it again and assay both the soluble solution and solids to find where is your desired protein ■ If it’s in the solid precipitated protein, we’ll go onto the next kind of purification ■ If it’s in the soluble protein, what we’ll do is increase the saturation (for example to 40%) then assay again, until you get the protein in the precipitate ■ If you do this well you can get an 80% purification - aka partially purified 2) Separation by charge (ion exchange chromatography): Use charged beads that are the size of grain of sand 6

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Ex: you have a DNA binding protein in the previous step ● Take a glass column, use a (-) charged beads (fill column half way up with this) ● Take the partially purified from solubility separation step 1 and put it over the bead ● Let liquid run through the column into a test tube. Once 1 mL of liquid is collected in the test tube, we change and start another fraction ● Buffer the fractions (to keep pH constant) and put a fairly low concentration of sugar in (KCl) ○ (-) charge proteins will elude out of the column first because of repulsion (early fraction aka 1-10 fraction) ○ (+/-) proteins come off ○ (+) charged proteins will stick on the bead due to attraction ■ Increase salt concentration with a strong electrolyte to out compete the proteins (+) charge - strong electrolytes will bind and (+) proteins will fall of bead and have to leave the column - around 1M KCl all (+) protein will be washed off ■ (+) charge proteins will elute out in late fractions ○ Now you have 100 test tubes with 1 mL of fraction, we assay all of them to see which has the desired protein ■ Hopefully only a few test tubes have the protein because it means that there was a big purification ■ The ones that do have protein will get pulled and move onto the next purification step 3) Separate by size/mass (size exclusion chromatography): use beads with pores instead of a charge - Large proteins cannot get into the pore, while small ones can - You place these into a tube, pour what you collected from step 2, collect fractions, and assay them to see which fraction contains protein - Large proteins will elute first due to them not being able to bind to the pores of the bead (early fraction) - The smaller the protein, the more time it’ll spend in the pore. These will elute last Picture of the graph I found on the internet and picture of bead

4) Affinity chromatography - Use a completely pure isolated protein and a molecule that specifically binds to the isolated protein - Types of molecules that you could use to bind to isolated protein 1) Antibodies - very specific, will only bind one protein - Can attach antibodies to beads, make a column out of them, put extract over it, and only protein you want will bind, and wash with salt to get pure protein off beads 2) Cofactor - non protein part of enzymes that are needed for enzyme activity. Usually very strongly and specifically bind to their specific protein 3) Substrate - Linked covalently to bead ● DNA binding protein - if you know specific DNA sequence use this - Make DNA, attach to a bead, run an affinity column, collect fractions, run assay on fraction ● RNA binding protein - same as DNA but with RNA - Make RNA, attach to a bead, run an affinity column, collect fractions, run assay on fraction (same as DNA but with RNA) 7

Molecular biology - Used for single proteins/single polypeptides Steps for Molecular biology 1) Clone the gene of interest 2) Subclone the gene into what are known as expression vectors ● Expression vector are usually are plasmids (small circle of nucleotides) ○ Could have a lot of plasmids per cell, which is nice when you have a lot of gene and want a lot of protein ● Subclone gene behind a strong promoter so you can make lots of copies per gene ○ You’ll have millions of copies and can make 30-40% of total proteins your protein ● When subcloning you’re also going to tag your protein ○ Ex: 6 histidines on the N terminus or Carboxyl terminus - gene will produce these protein ■ Lyse open cell, put over affinity chromatography column with beads that have nickel on it, and only things that will bind to those are going to the be the tagged 6 histidine protein Lecture 3 - 2/17 - What to do with pure proteins Let's assume you have a isolated protein - First, you want to know the amino acid sequence of the protein - Done via mass spectrometry - this very accurately measures the mass of a molecule ● Measuring biological samples with mass spec is relatively new because steps to prepare for mass spectrometry would typically destroy the proteins a) Need a gaseous ion to run mass spec b) Gentle methods were discovered to produce this gaseous ion from biological proteins Gentle methods 1) Electrospray a) Put protein in solution b) Pump through glass capillary c) Create a fine mist of the solution d) Put a high charge gradient (high voltage) across the mist - if this is done correctly, you will get the protein into the gaseous stage as positively charged ion e) Spray into mass spec to measure it

2) MALDI (Matrix Assisted Laser Desorption Ionization) a) Make a solution of your protein into a gel b) Hit it with a laser of the proper frequency - if you do this right you get gaseous ions 3) Protein sequencing center a) They break your protein up into fragments using trypsin, measure the individual masses of the fragments, and put them together ● Trypsin is a protease that breaks peptides binds ○ Specific for only peptide bonds only after positively charged residues (aka Lysine and Arginine) ○ Ex: “Say you have a protein with 5 arginine residues and 5 lysine residues, and you completely digest it with trypsin, then you’re going to have 11 fragement (I think you include trypsin as a fragment?) b) You make the fragments into a gaseous ion via maldi or electrospray and figure out their mass ● This doesn’t tell you anything directly - aka it doesn’t tell you the amino acid sequence 8

c) Need compare the mass to a database and have computational ability - Computer scans all the reading frames and determines the amino acid sequence ● This is helpful if your protein has been studied before ● If you’re doing a species that hasn’t been sequenced yet then you’re out of luck ● Let’s assume (c) worked and the database was able to give results. What does that mean? ○ Computer could tell you a whole lot or nothing at all d) If you get amino acid sequence do computer analysis - Ask 2 questions: 1) Has the same protein ever been worked on before? 2) Are there other related proteins in other species that have been worked on and function of those proteins has been discovered? (homology searches) ➔ If there are no homologies to your protein you can search for motifs ◆ Google says “Motif is a pattern of defined amino acid residues that are important for protein function and are located within a certain distance from each other” ◆ In Roe...