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by: Alice Hsu

BIO 12 WEEK 2 NOTES Biology 12

Marketplace > Dartmouth College > Biology > Biology 12 > BIO 12 WEEK 2 NOTES
Alice Hsu

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this week we covered the protein shape and function and experimental approaches.
Cell Structure and Function
Natasha Grotz
Class Notes
Biology, Cellular and Molecular Biology, Proteins, Protein function, experimental
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This 5 page Class Notes was uploaded by Alice Hsu on Saturday September 24, 2016. The Class Notes belongs to Biology 12 at Dartmouth College taught by Natasha Grotz in Fall 2016. Since its upload, it has received 2 views. For similar materials see Cell Structure and Function in Biology at Dartmouth College.

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Date Created: 09/24/16
BIO 12 WEEK 2 NOTES MORE FLUORESCENT MICROSCOPY: How to tell if a fusion protein is fully functional?  Complementation assay: cross a mutant and normal and see if it expresses in the F1 gen.  Use immunofluorence and compare. Possible problems to GFP:  DNA must be introduced into cell (what if it is rejected????)  Fusion may change the function and localization FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)  Measures the change in proximity of cellular compounds labeled with fluors. o If the fluors are close enough (5-10 nm) in distance and emission/excitation spectrum. o Exciting one will then cause the other to emit. Fluorescent dyes can be used to monitor molecules—heat maps are used to see gradients LASER CONFOCAL FLUORESENCE MICROSCOPY  Eliminates out of focus light via pinhole aperture  Laser excites fluorophores  This method means that we can only see one focal length. o Thus, multiple focal lengths (z-sections/thin, optical sections) are taken and pieced together to create a 3D image of a much thicker specimen.  Can be used for immunofluorescence or fusion proteins Direct interaction: literally touching. ELECTRON MICROSCOPY  Transmission EM (TEM): Electrons transmit through specimen  Scanning EM (SEM): electrons bounce off specimen (image is obtained indirectly)  Where light microscopes use glass lenses, EM uses metal plates.  Since they depend on electron wavelength, the resolution is much better (about 1-5 nm for TEM, 5-10 nm for SEM) Prepping the sample for TEM:  Fixed sample  Embed sample in plastic  Sample very thinly sectioned  Stained with heavy metals  Image (in SEM, the sample is dehydrated and is coated with carbon and gold instead of heavy metals to ensure that the electrons bounce back) More about staining  The metals bind to the proteins and membranes, which become dark details  Negative staining: the grid is stained while the specimen is not stained  Metal shadowing: metal is blown from one side of the grid to get a 3D image  Freeze fracture/etch: o Separates membrane o Sublimation of ice layer o Shadow of metal-carbon replica How to differentiate between microscopes  Do you want to see color?  Do you want to see in 3D?  What scale is your sample at? PROTEIN STRUCTURE AND FUNCTION Proteins are the workhorses of the cell Amino Acids: Zwitterion: amino acid at a neutral pH; the carboxylic acid is deprotonated and the amino group is protonated  (Nonpolar) Hydrophobic amino acids: o R-group consists of C-H skeleton o Forms inner core of soluble proteins away from aqueous medium o Associates with lipid bilayer  (Polar) Hydrophilic amino acids: o R-groups are neutral but can form H bonds because of a partial charge due to dipoles  Polar -charged amino acids: o R-groups act like acids/bases, which tend to be fully charged under neutral conditions o Forms ionic bonds  Special cases: o Glycine: small R-group (-H), which gives it a lot of environmental flexibility o Cysteine: sulfhydryl group, which makes disulfide dimers with other cysteines o Proline: R-group is a rigid, structural rings that introduces a kink in the protein, disrupting order Polypeptides form via condensation reactions: Structure:  Primary: linear sequences of amino acids  Secondary: localized folding of amino acid o Alpha-helix  Stabilized by H-bonds between C=O and N-H backbones about 4 aa apart  R-groups stick out as a result  Very regular and favorable (unless proleine is involved)  Important for integral membrane proteins (like phospholipid bilayer) o Beta-strand: becomes a beta-sheet  Tertiary: overall 3D conformation o Beta-sheet:  R-groups alternate to make pleated structures  H-bonds between C=O and N-H for stability  Strands can be arranged in parallel (same direction) or anti-parallel (opposite directions) o Stabilized by various forces:  Covalent:  Disulfide bonds  Non-Covalent:  Ionic  H-bonds (also make transient interactions possible)  Van der Waals  Hydrophobic bonds (which are especially important for hydrophobic proteins in aqueous environments who want to minimize interaction)  Quaternary: multiple tertiary structures bonded together A polypeptide may have a combination of second degree structures. Protein function  Proteins function as a community  Shape matters for a protein to function properly  Domain: a structural molecule within a protein with a distinct function that is often conserved between proteins  Ionic associations or covalent modifications can change the 3D structure of a protein and regulate how the proteins interact with each other o See: Phosphorylation, a type of post-translational modification located in sarcomere EXPERIMENTAL APPROACHES Case study: mitochondrial protein MPV17 We detected this protein with the help of immuno-gold TEM. The mutation leads to liver failure. 1) We must purify the protein, which involves the stepwise removal of contaminants. All proteins have a characteristic size, shape, charge, hydrophobicity, and post-translational modifications. a. Questions to ask: i. What cells should I use? ii. What process should I pick to purify the cells? iii. What type of cell is likely to have my protein? iv. Does it involve cell-cell communication or tissue? b. Working with cultured cells: i. More homogenous ii. Controllable conditions iii. Can isolate single cells iv. Primary cell cultures are established from animal tissues v. Some “transformational cells” (e.g. cancer cells) are immortal and can be used to form a cell line. Note: not all cells can be studied in cultures c. Cell lysis: i. Sonification ii. Force cells through small holes + centrifugation: spinning sample iii. Detergent (for permability) at various speeds iv. Tissue homogenizer ( a bullet blender to separate for tissue samples) components by density Equilibrium density centrifugation: components will sediment in a density gradient until they reach their own buoyant density  Useful for: o Separating organelles from each other o Separating membranes 2) We have localized MPV17 to the inner membrane of mitochondria. How do we extract an integral membrane protein? a. Detergents! (they are amphipathic and vary in gentleness i. SDS: less gentle. Unfolds proteins ii. Triton-X100: more gentle. Retains forms. b. Column chromatography: separate the components by running a sample through a matrix. i. Ion exchange: separating proteins by charge. 1. The ion that matches the solvent charge will run faster. 2. Changing the pH of the eluent helps the other charged ions to release without affecting the solid matrix. You can also add salt. ii. Gel filtration iii. Affinity The relationship between protein charge and pH is direct. The more acidic the protein is the lower the pH.


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