Kinetics Review. Tonight at 7 PM Phys 204 We will do two problems on the board (additional ones than in the problem sets)

Transcription

1 Quiz 1

2 Kinetics Review Tonight at 7 PM Phys 204 We will do two problems on the board (additional ones than in the problem sets) I will post the problems with solutions on Toolkit for those that can t make it 2

3 Dr. James Watson in the News Again 3

4 Review H146 Fβ D94 C K40 Fα 4

5 Hemoglobin 5

6 Methods used to investigate protein structure and function Need to prepare protein of interest for structural studies and most functional studies Protein purification SDS PAGE gel electrophoresis Proteomics (high-throughput analysis of protein mixtures (e.g., from cell extracts) 2D SDS-PAGE Mass spectrometry 6

7 Why purify proteins? Purified proteins can be used to: Study enzymatic functions and enzymatic regulation Study protein interactions Produce antibodies Perform structural analysis by x-ray crystallography and NMR spectroscopy 7

8 How to purify proteins? Biochemists exploit he ways individual proteins differ from one another, such as solubility, size, or charge. Specific binding 8

9 Size-exclusion chromatography 9

10 Size-exclusion chromatography Absorbance at 280 is used to identify protein-containing fractions. You can also perform an enzyme specific assay. 10

11 Ion-Exchange chromatography - - If ph mobile phase =7.2 Then charge of the proteins: (-) (-) () () Anion exchange column matrix is charged Cation exchange column matrix is - charged 11

12 Ion-Exchange chromatography Na - Na - - Na - - Na - Na Na - Na Na Na Cl- Cl- Cl- Cl- Cl- Cl- Increased salt concentration 12

13 Affinity chromatography Makes use of specific binding interactions between molecules 1- Incubate crude sample with the immobilized ligand 3- Elute 2- Wash away non bound sample components from solid support 13

14 Affinity chromatography Commonly used affinity columns: Ni2 binds to poly Histines (example 6xHis) Protein genetically fused with tag Specific antibodies (anti-flag tag) glutathione binds to GST Protein genetically fused with fusion Possible elution strategies: ph Ion strengh Denature Competitor ligand or analog 14

15 Ni-NTA columns The Ni-NTA resins have a high affinity for 6xHis-tagged proteins NTA has a tetradentate chelating group that occupies four of six sites in the nickel coordination sphere How would you elute? 15

16 Evaluating protein purity and molecular weight with SDS-PAGE gel SDS is a negatively charged detergent that typically denatures proteins 16

17 Electrophoresis of protein sample on a polyacrylamide gel (PAGE) a polymerized, highly cross-linked matrix All protein/sds micelles are negatively charged; therefore migration is solely based on molecular weight s-s SDS, heat proteins with SDS - Reducing gel reducing agent present disulfides do not remain intact Non-reducing gel no reducing agent disulfide bonds maintained 17

18 An example of an SDS-PAGE gel of a purification of a His-tagged protein kd

19 Proteomics Analysis of whole proteome or a subset of the proteome a mixture of proteins Secreted proteins Nuclear proteins Membrane proteins Process many samples quickly (high-throughout) 19

20 Examples of Proteomics 2D Gel Electrophoresis Activity Based Probes Protein Microarrays 20

21 2D-SDS PAGE gel The first dimension (separation by isoelectric focusing) - gel with an immobilised ph gradient - electric current causes charged proteins to move until it reaches the isoelectric point (ph gradient makes the net charge 0) 21

22 Isoelectric point (pi) Separation by charge: Stable ph gradient Low ph: Protein is positively charged High ph: protein is negatively charged At the isolectric point the protein has no net charge and therefore no longer migrates in the electric field. 22

23 2D-SDS PAGE gel The first dimension (separation by isoelectric focusing) - gel with an immobilised ph gradient - electric current causes charged proteins to move until it reaches the isoelectric point (ph gradient makes the net charge 0) The second dimension (separation by mass) -ph gel strip is loaded onto a SDS gel -SDS denatures and linearises the protein (to make movement solely dependent on mass, not shape) 23

24 The Second Dimension of a 2D SDS-PAGE gel 24

25 2D-gel technique example 25

26 Protein Identification by MS Spot removed from gel Fragmented using trypsin Spectrum of fragments generated Library MATCH Artificial spectra built Artificially trypsinated Database of sequences (i.e. SwissProt) 26

27 Advantages vs. Disadvantages Good resolution of proteins Detection of posttranslational modifications Not for hydrophobic proteins Limited by ph range Not easy for low abundant proteins Analysis and quantification are difficult 27

28 Proteomics and Function Activity based probes Labeled proteome activity profile. Representative in gel fluorescence analysis of the secreted proteome (labelled with fluorescent pigment) derived from invasive MUM-2B and non-invasive MUM-2C human melanoma cancer cells. Enzyme activities selectively associated with either invasive cells (for example, urokinase) or non-invasive cells (for example, sialic acid 9-O-acetylesterase or SAE) are noted 28

29 Protein microarrays DNA microarrays assay mrna levels; however, a correlation between mrna levels and protein expression is not always observed 29

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34 Protein Structure Conformational changes Structure Based Drug Design 34

35 X-ray crystallography 35

36 NOESY spectrum 1 H chemical shift (ppm) 1 H chemical shift (ppm) NMR Provides distances between atoms Put all the distances together and calculate a structure 36

37 Determining structure of two states of the protein Hexokinase Glucose ATP Glucose-6-P ADP Open unbound substrate Closed bound substrate 37

38 Molecular Dynamics Computationally simulate the motion (solve equation of motion) of biomolecules in silico 38