Samantha s SI Sheet Week 4 Protein Toolbox and DNA Basics Things to Know Purpose of dithiothreitol and iodoacetate carboxymethylate sulfhydryls DNA is awesome storage understand magnitude to which DNA can store hard drives etc Resolution of X ray crystallography different n lengths Better resolution more detail Structures to recognize Edman reagents Merrifield reagents denaturants Recognise know names do not need to be able to draw structure 1 What are the two factors that 2D gel electrophoresis separates on a Charge isoelectric focusing separating on pI pH at which protein is neutral b Mass separate by mass chain length through SDS page 2 What happens when you treat a protein with 6M HCl for 24 hours What information can we collect and how a Hydrolyse protein into tiny bits of amino acids It is complete hydrolysis so all amino acids separate from each other Now you have a solution of amino acids Use chromatography and use elution volume to figure out how much aa1 aa2 aa3 etc you have Use ninhydrin and fluorescamine used to visualize and thus measure amino acids Based on absorbance of sample you can figure out how much of which amino acid you have b BUT you do not know the sequence of the amino acids 3 Why can Edman degradation give you only up to 30 50 amino acids of information How do we adapt to this problem How many amino acids of information do you need to uniquely identify a protein a Chop off one piece at a time from N terminus phenylisothiacyanate recognize creates a visual PTH molecule that can be seen By measuring absorbance you can figure out what amino acid the protein has at that end you chopped off After 30 50 amino acids the graph is too messy inaccurate due to too many peaks so you cannot go past this for Edman degradation to read the graph b Fix by chopping protein into fragments but you don t know the fragment order c Must do at least 2 sets of Edman degradation created at different points use different kinds of proteases cyanobromigen or trypsin d There are 25000 proteins in human body 1 20 n 1 25 000 Therefore n 4 amino acids 4 Compare and contrast using antibodies with Mass Spectroscopy aka MALDI TOF a Use both for identifying proteins b Mass spectroscopy how long it takes particles to get from one side to the other Take sample bombard with laser makes ions which travel at different speeds depending on size Given graph of ion fragments known patterns you can get the sequence of amino acids c Antibodies cannot get you sequence good for quick identification often in clinical settings of proteins i Mono vs polyclonal antibodies can have multiple antibodies responding to the same protein since antibodies bind to small parts of the protein Polyclonal multiple different antibodies bind to the protein 5 Order the steps of solid phase protein synthesis What molecules reagents are involved a Protect C terminal amino acid synthesize in C N direction b Attach amino acid to resin by attacing it to solid face increases efficiency yield CF3COOH c Deprotect amino terminus of C terminal amino acid Do this with d Add protected and activated amino acid residue Protect N terminal with tBoc activate C terminal with DCC e Deprotect amino terminus f Repeat D and E g Release peptide from resin 6 Label whether the following statements are true for NMR X ray crystallography or both a Used to determine 3D protein structure BOTH b Requires protein to be in solution NMR c Gives a picture with shades and dots XRAY d Uses electron location to give shape information XRAY e Can focus on a specific proton to see how it affects the rest of the protein NMR 7 Draw an A T nucleotide base pair a Look in PP or notes for nucleotide structures Remember that strands run antiparallel 8 What would be different in the drawing if this was RNA a There would be uracil instead of thymine Ribose would be used instead of deoxyribose and ribose has a 2 OH and is less stable can hydrolyze phosphodiester bond Also RNA tends to be single stranded 9 Why does RNA sometimes have tertiary structure a If RNA wants more functions it needs to have more structure than just a single strand i Different functional RNAs include mRNA tRNA rRNA siRNA ii Structure offers fuynction One common motif in RNA is a stem etc loop 10 Biochemistry Analogies Ex Professor Lecture TA Lab a Protein N C DNA 5 3 b Amide Protein Phosphodiester DNA bonds c B DNA 10 alpha helix 3 6 bp turn