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C.5 Lecture notes Primary structure.docx

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Biological Sciences
Rongmin Zhao

Primary structure - AA sequence Secondary: local spatial arrangement of a polypeptide backbone atoms through H-bond interactions Tertiary 3D structure of entire chain Quaternary: spatial arrangement of diff polypeptide chains in a protein • 1 polypeptide can be cleaved to form more than 1 protein 1 gene can be transcribed into different mRNA's 1 mRNA can be translated into multiple proteins PROTEIN PURIFICATION • Solubility: - salting out: increase ionic strength increase solubility (salting in), then decrease (salting out) • Ionic charge: ion exchange chromatography electrophoresis isoelectric focusing • Polarity: hydrophobic interaction chromatography • Size: -gel filtration chromatography -SDS-PAGE • Binding specificity: affinity chromatography • Only functional protein All purification methods must be based on the properties of the target proteins If the properties of the protein is unknown, different strategies have to be tried. HOW ARE PROTEINS ISOLATED AND PURIFIED FROM CELLS? • 1000's of proteins in cells can b separated and purified on basis of size and electrical charge • Proteins tend to be least soluble at their pI • increasing ionic strength at 1st increases solubility of proteins (salting-in, then decreases it (salting-out) SALTING IN - decrease [salt] cause protein to precipitate (solubility decreases)? These 2 are opposite processes SALTING OUT - increasing [salt] cause protein to precipitate • High concentration of salt added to protein solution to reduce solubility of protein • Target protein will precipitate at a specific salt concentration, at which the other proteins will remain soluble • Common salt: (NH4)2SO4 -> high solubility and not significantly affected by temperature • High concentration of (NH4)2SO4 will take away H2O molecule originally around the protein molecule -then electrostatic interactions bn protein molecules make proteins precipitate • Initially all are soluble in buffer, add salt to solution, unwanted protein precipitates (mb remove by centrifugation) -supernatant on top *proteins are least soluble at pI, it's a pH thing COLUMN CHROMATOGRAPHY (general term) Types: 1. Ion exchange 2. Hydrophobic 3. Gel-filtration 4. Affinity chromatography • Use a column, fill with matrix, apply protein mixture, let protein bind, wash away bound or unbound protein, elute • Protein has diff affinity, elute using this • Properties define the types above -alternative way to column chromatography is the Batch operation: -the binding matrix is placed in a centrifugation tube, the binding, washing and elution of protein is conducted by sequentially addition and removal of solution manually -extremely useful for small scale operation ION EXCHANGE CHROMATOGRAPHY Binding: low salt concentration Washing: low salt -> wash away unbound proteins Elution: high salt If matrix negatively charged, then +'ively charged group will remain Decrease pH, proton will bind likely -> +'ive charge At the end, can add high salt to compete with protein to make precipitate Cation exchange: CM (carboxymethyl): Matrix-CH2-COO- Mono S ®: matrix-O-CH -CHO2-CH -O-CHOH2CH -SO 2 3- Anion exchanger: DEAE (diethylaminoethyl): matrix-CH2-CH2-N-H(CH2CH2)2 Mono S ®: matrix-O-CH -CHO2-CH -O-CHOH2CH -N-(CH3)3 2 Matrix: usually inactive polymers *for all protein purification, need a buffer system GEL FILTRATION CHROMATOGRAPHY - separate based on size • A gel filtration chromatography column: beads filter purely on size, smaller molecules enter beads • Larger molecules are excluded from gel beads and emerge from column sooner than smaller mole's, whose migration is retarded b/c they can enter the beads SIZE-EXCLUSION CHROMATOGRAPHY/MOLECULAR SIEVE/GEL FILTRATION Holes inside beads. Proteins get inside and come out continuously, smaller get stuck longer (delayed). It’s a matrix inside bead like puzzle -large bypass the beads • Usage of gel-filtration: • Purification of protein from contaminant proteins • Determination of protein size (using molecular weight standard and assuming the globular size of protein) • Desalting • Usually last step of protein purification AFFINITY CHROMATOGRAPHY 1. Binding, washing and elution involved 2. Commonly used affinity tags: 6xHis: binding to Ni2+ and eluted with imidazole FLAG ® tag: peptide: DYKDDDDK. Binding to antibody and eluted with FLAG peptide proteinA: ~20kDa protein, binding to IgG and eluted with low pH • Does not depend on exactly structure of protein, can even denature protein -> still work • Easily elute protein using imidazole solution, side chain of histidine is imidazole, compete with protein Process to make more pure after each step 1. Crude extract 2. Salt precipate 3. Ion exchange chromatography 4. Molecular sieve chromatography 5. Immunoaffinity chromatography -> after this step, 1000x more pure PROTEIN ANALYSIS 1. Analysis of chemical and physical properties of target proteins: MW, AA composition, amino acid sequence, pI, molecule size and shape etc. 2. Analysis usually associated with purification -itself can be a purification process 1. A focus will b given on protein primary sequence determination ANALYSIS OF PROTEIN BASED ON SIZES (SEPARATION OF PROTEINS) SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) Reducing agent: DTT (Dithiothreitol) Beta-mercaptoethanol • SDS (sodium dodecyl sulfate) -> a strong ionic detergent • After binding to SDS, all proteins have the same charge:mass ratio so that separation is only based on MW by using networked polyacrylamide gel • 1 SDS molecule binds to 2 amino acid residues •Stain with Coomassie blue •Make a plot of protein mobility vs. log of MW of individual peptides -roughly determine exactly size of protein 2D GEL ELECTROPHORESIS •Macromolecules are 1st separated according to charge by isoelectric focusing in a tube gel •The gel containing separated molecules is then placed on top of an SDS-PAGE slab, and mole's are electrophoresed into the SDS-PAGE gel, where they are separated according to size •The individual spot can be extracted for other purposes like sequencing, mass spec etc. •The individual spot is not necessarily corresponding to only 1 polypeptide •Separates LEC 6 - PROTEIN AMINO ACIDS COMPOSITION Anion exchange for -'ively charged protein, Could use cation exchange as well, protein wont bind, will come out in washing stage -> negative selection HOW IS AA HYDROLYSATE FORMED FOR DETERMINATION OF AA COMPOSITION? •Either purified protein or protein mixture •Acid hydrolysis liberates the AA of a protein, into individual AA's •Note that some Aas are partially or completely destroyed by acid hydrolysis •Chromatographic methods are used to separate the amino acids •The AA compositions of diff. proteins are different AA HYDROLYSATE TREATED WITH PITC (phenylisothiocyanate) Some of the amino group will be deprotonated (pKa >9) Each AA goes through this cycle, f
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