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Lecture 3

BIOC12Fall2012 Lecture Week 3 Notes

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Biological Sciences
Rongmin Zhao

BIOC12Fall2012 Lecture Week 3: Protein primary structure and analysis (Chapter 5) Protein Structures o Primary (1°) structure: protein amino acid sequence o Secondary (2°) structure: the local spatial arrangement of a polypeptide backbone through H-bond interaction  without considering its side chains o Tertiary (3°) structure: 3D structure of the entire polypeptide chain o Globular conformation gives the lowest surface-to-volume ratio, minimizing the interaction of the protein with the surrounding environment o Quaternary (4°) structure: spatial arrangement of different polypeptide chains in a protein  not all proteins have a quaternary structure  subunit conformation (subunits of tertiary structure come together to form this quaternary structure) o Secondary and higher order structures are determined by non covalent forces One Polypeptide can be cleaved to form more than One Protein o The primary sequence of amino acids  translated in the ribosome o One long polypeptide chain can be cleaved to form more than one proteins o Ex: ubiquitin + ribosomal protein L40e form one polypeptide chain in S. cerevisiae  the polypeptide chain form ubiquitin which covalently conjugated to proteins thus marking them for degradation by the proteasome & L40e is a subunit of the ribosome large subunit of S.cerevisiae Peptide vs. protein o By convention, only those polypeptides at least 40 amino acids long are called proteins  the shorter ones are peptides, dipepties, tripeptides, etc. o A peptide longer than 40 amino acids can fold to a stable shape to carry out particular functions 2+ 2+ o Proteins can bind metal ions such as Zand Mg , which confer important functions o The occurrence frequencies of the 20 standard amino acids in protein are different  leu, ala, gly, ser, val, glu are abundant, while trp, cys, met, and his are rare Strategies to purify proteins o o all purification methods must be based on the properties of the target proteins  properties of proteins come from their side chains o if the properties of the protein are unknown, then different strategies must be tried How are proteins isolated and purified? o Thousands of proteins in cells can be separated and purified on the basis of size and electrical charge o Proteins tend to be least soluble at the pI o Increasing ionic strength at first increases the solubility (salting in),then decreases it (salting out) o Figure: the solubility of most globular proteins is influenced by pH and ionic strength o  they become more soluble as the ionic strength is increased 1 o Green curve  At low pH, the solubility is high, then it becomes lower as it reaches/come close to the isolelectric point, and then it becomes soluble again as the pH increases. o Blue curve  4 M salt concentration – the protein has very low solubility in high salt concentration o Pink curve (1mM salt concentration) – protein is soluble as long as it is not near its isoelectric point o There is an optimum salt concentration, which increases solubility. (bell curved graph – turn direction at a certain salt concentration) o Principle of protein salting out o Salting out procedure causes the target molecule to precipitate out of solution o High concentration of salt is added to protein solution to reduce solubility of the proteins o The target will precipitate at a specific concentration, at which the other proteins will remain soluble o Common salt: (NH4 2SO4 has high solubility in water and is not significantly affected by temperature o High concentration of (NH ) SO will take away the water molecules that are originally around the protein molecules 4 2 4  then electrostatic interaction between protein molecules will make the proteins precipitate Column chromatography o There are 4 kinds: ion exchange, hydrophobic, gel filtration and affinity chromatography o Based on differential interaction of target protein and unwanted proteins in the matrix o Proteins come out at a different times or different elution volumes o an alternative way to column chromatography is the BATCH operation o the binding matrix is placed in a centrifugation tube o the binding, washing and elution of protein is conducted by sequentially adding and removing solution manually o extremely useful for small scale operations o Ion exchange chromatography o o binding = low salt, washing = low salt, elution = high salt 2 o cation exchanger: negatively charged matrix so it can bind positively charged molecules  CM (carboxymethyl) and Mono S o anion exchanger: matrix is positively charged so it can bind negatively charged molecules  DEAE (diethylaminoethyl) and Mono Q o ** the matrix is usually made up of inactive polymers Gel filtration chromatograph o o can be used to separate molecules based on size o large molecules are excluded from the gel beads and so emerge sooner from the column o the smaller molecules can enter in and out of the gel beads, so migration to the end is slower o Size- exclusion chromatography o usage of gel filtration o purification of protein from contaminant proteins o determination of protein size  by using MW standard and assuming the globular size of the protein o desalting  usually last step of purification 3 Affinity chromatography o you have to choose 1:1 binding pairs o binding =, washing and elution steps are involved o commonly used affinity tags are 6xHis (bindin to Ni2+ and eluted with imidazole) o FLAG tag peptide (DYKDDDDK)  binds to antibody and eluted with FLAG peptide o Protein A: 20kDa protein, binding to IgG and eluted with low pH o The yield of a typical protein purification process 4 o o more than one step required to purify o the higher the specific activity , the higher the purity of the sample o that is why the more steps undertaken, the higher the purity of the sample Protein analysis o analysis of a chemical and physical properties of target proteins  MW, amino acid composition, amino acid sequence, pI, molecule size and shape etc. o analysis is usually associated with purification  analysis itself can be a purification process o focus will be given on protein primary sequence determination Analysis of protein based on sizes (separation of protein) SDS- polyacrylamide gel electrophoresis (SDS-PAGE) o DTT is the reducing agen which will reduce disulfides bonds  B-mercaptoethanol can also be used to serve same purpose o SDS is a strong ionic detergent o After binding to SDS all protein have the same charge to mass ratio so that separation is only based on MW by using polyacrylamide gel o 1 SDS molecule binds to 2 A.A o larger polypeptides will move slower through the gel matrix o the gel is stained with coomassie blue so you can see the protein and how much it has run o the more purification steps done, more protein would show up in the bands in gel because the specific activity will increase  more of the target molecule will be present in the same volume of the sample o a plot of the protein mobility vs log of MW can be plotted and peptides identified based on their MW o 2D gel electrophoresis 5 o o macromolecules are first separated accoding to charge by isoelectric focusing in a tube gel (left to right separation based on pI) o then molecules are electrophores into the SDS-PAGE gel where they are separated by charge o example: of pI of protein is 5.6 then at pH of 4 it will be posit
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