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Lecture 4 learning goals Describe the key factors that determine protein structureExplain why the structure of proteins is importantDescribe mechanisms that regulate how proteins are controlledTechniques used to study proteinsHow do they work?What do the results look like? How do you interpret them?Which techniques separate proteins based upon known characteristics of those proteins?

1Online Protein Introduction VideoClicker Question Review!!!Protein Structure

Primary sequence contains a sequence that will predict the structure Secondary struvtutr you can see that there is certain amino acids that allows the stryctyre t3Protein Structure

Secondary StructureTertiary StructureThe n terminus starts with beta strands the direction of the arrow points to the C terminus

4Secondary Protein Structure

alpha helixbeta-pleated sheetnot all regions of proteins are organized into these structures. There are regions within proteins that are disordered or flexible.largely a result of interactions between amino acid backbones in the polypeptide chain5Secondary Protein Structure

alpha helixbeta-pleated sheetnot all regions of proteins are organized into these structures. There are regions within proteins that are disordered or flexible.largely a result of interactions between amino acid backbones in the polypeptide chain

Many proteins are embedded in membraneThe method of protein integration into the membrane is not spontaneous. We will learn more about this in Chapter 15.

Bonds that Maintain Protein Structures

non-covalent bondscovalent bondsside chainsTetiary structure uses non coovalent interaction 8Hydrophobic Forces Drive Protein Folding

The hydrophonbic goes inwards\NONPOLAR = hydrophobic

9amino acids that are commonly modified:Post-translational ModificationsDisulfide bonds between cysteines

Phosphorylation onserine, threonine, or tyrosineCytosol is a highly reducing environmentMany extracellular proteins are held together by disulfide bonds

Disulfide bonds will stabilize structure in protein post-translational modificatiosn Cytosol proteins dont contain disulfide bonds ***** Phosphor is the addition of a phosphate group10amino acids that are commonly modified:Post-translational Modifications

Glycosylation on asparagine, serine, and threonineUbiquitination on lysine

N-linkedO-linkedGlycosylation= sugar

11Some proteins have multiple domains

Fibronectin (ECM) has 4 successive domains of the same type

Src protein has 4 distinct domainsDomain: different regions of the polypeptide chain fold independently

Sh2 domain bind to phosphoraled tyrosine residuesSh3 nignd to proline rich sequences We can guess the function of a protein if we look at the doamian 12Proteins can share domain structure

may be well-conserved regions appearing in many proteins/organismswhich protein might this be?NC13Different Visual Representations of Protein Structure4 Models:Backbone (chain)Ribbon modelWire modelSpace filling model

We should guess what the thing is by looking at sturcuture

14Quaternary Protein Structure =Multi-subunit proteins dimers, trimers, tetramers, etc..Usually linked by non-covalent bonds and/or hydrophobic surfacesCan be made up of hetero- or homo-associations

Not all proteins have quaternary protein Homotypic two of same type come togetherHeterotopic 2 of the different type come together

15Protein Folding can create Binding Sites

Shape and position is important.Having multiple non covalent

Changing a single amino acide always always always changed the primary structure It depends on the amino acid if it changes the secondary tertiary or cell function

16protein case study:sickle cell anemia NORMALLY, the tetramer of hemoglobin subunits are held together by: ionic bonds, hydrogen bonds, and hydrophobic interactions

when O2 binds to hemoglobin, it changes the tertiary and quaternary structure of the complex (conformational change)

hemoglobin schematic from wikipedia commonsprotein case study:sickle cell anemia NORMALLY, the tetramer of hemoglobin subunits are held together by: ionic bonds, hydrogen bonds, and hydrophobic interactions

when O2 binds to hemoglobin, it changes the tertiary and quaternary structure of the complex (conformational change)

hemoglobin schematic from wikipedia commons

How Proteins are ControlledProtein levels (gene expression, protein production, protein degradation)Confining Proteins to Subcellular compartmentsAlter Protein Conformation (and thus its function)Most proteins are allostericHave two (or more) slightly different conformationsProtein activity can be regulated by shifting between these conformationsBinding of a regulatory protein/moleculeCovalent modifications on ProteinsPhosphorylationAcetylationUbiquitination19How Proteins are ControlledProtein levels (gene expression, protein production, protein degradation)Confining Proteins to Subcellular compartmentsAlter Protein Conformation (and thus its function)Most proteins are allostericHave two (or more) slightly different conformationsProtein activity can be regulated by shifting between these conformationsBinding of a regulatory protein/moleculeCovalent modifications on ProteinsPhosphorylationAcetylationUbiquitination

How are proteins regulated Protein levels how much protein is presence whether it is abundant or scarce Like lysosome Changing the proteins shape can control the proteins the functional site is on one site and the functional binding site is on another site

20How Proteins are ControlledProtein levels (gene expression, protein production, protein degradation)Confining Proteins to Subcellular compartmentsAlter Protein Conformation (and thus its function)Most proteins are allostericHave two (or more) slightly different conformationsProtein activity can be regulated by shifting between these conformations1) Binding of a regulatory protein/molecule2) Covalent modifications on ProteinsPhosphorylationAcetylationUbiquitination

- 21How Proteins are Controlled

Protein levels (gene expression, protein production, protein degradation)Confining Proteins to Subcellular compartmentsAlter Protein Conformation (and thus its function)Most proteins are allostericHave two (or more) slightly different conformationsProtein activity can be regulated by shifting between these conformations1) Binding of a regulatory protein/molecule2) Covalent modifications on ProteinsPhosphorylationAcetylationUbiquitinationGTP has a p that GDP doesnt have GTP is the active form. GDP is the inactive form GAP regulates how long the GTP is actibeve

22How Proteins are ControlledProtein levels (gene expression, protein production, protein degradation)Confining Proteins to Subcellular compartmentsAlter Protein Conformation (and thus its function)Most proteins are allostericHave two (or more) slightly different conformationsProtein activity can be regulated by shifting between these conformationsBinding of a regulatory protein/moleculeCovalent modifications on ProteinsPhosphorylationAcetylationUbiquitinationImage from: http://biomatics.org/index.php/Acetylation

How Proteins are ControlledProtein levels (gene expression, protein production, protein degradation)Confining Proteins to Subcellular compartmentsAlter Protein Conformation (and thus its function)Most proteins are allostericHave two (or more) slightly different conformationsProtein activity can be regulated by shifting between these conformationsBinding of a regulatory protein/moleculeCovalent modifications on ProteinsPhosphorylationAcetylationUbiquitinationImage by: Jennifer Kowalski (Butler University)mass spectrometryDetermines protein sequence or identityProteins often first digested with trypsinMeasure mass and determine chemical species within fragments

Composition and Structure can be determined for purified proteins

25Mass spec- to determine the sequence or identify a protein. Hit protein with electrons to split apart some molecules, these molecules are then separated by mass/chargeXray Crystallography- to determine 3 structure of proteins

What types of proteins or molecules are in your sample Should know protease

Composition and Structure can be determined for purified proteins

x-ray crystallographyUses crystals of purified proteins and determines (up to) tertiary structureBombard crystal with X-rays and then collect diffraction information26Xray Crystallography- to determine 3 structure of proteins

Protein structure Need to form chrystals from your proteins ( some proteins are okay with being crystalls Predict the tertiary structureXray have shorter rays compared to normal light

nuclear magnetic resonance (NMR) spectroscopyUses small-sized purified protein and determines (up to) tertiary structureIncubate protein solution in magnetic field and collect information about positioning of hydrogen nuclei ProteinsolutionNMR spectrumComposition and Structure can be determined for purified proteins

NMR Magnet27NMR depends on the physical properties of particular atoms to respond to magnetic field

You can predict where the molecules are located

Composition and Structure can be determined for purified proteinsSo how do we purify a protein?Break open cellsSeparate proteins from other cellular components (nucleic acids, lipids)Separate your protein of interest away from all other proteins within the cell

28This is importantRupturing the plasma membrane to release cell contentsHomogenization: breaking open cells

29Centrifugation: separating components within homogenant

Differential CentrifugationKnow this 30

chromatography to separate proteins based on some property of the amino acids/functionpart of purification process

Chromatography: separating/isolating proteins312 phases:Mobile phase- solvent that is moving (includes proteins of interest) Immobile phase- matrix that solvent moves through. Contains sites that proteins can bind to (or be trapped within) to slow down their movement. Greater interaction between the protein and the matrix, the longer it takes to move through the matrixchromatography to separate proteins based on some property of the amino acids/functionpart of purification process

several flavors:ion exchange gel filtration(affinity)Chromatography: separating/isolating proteins322 phases:Mobile phase- solvent that is moving (includes proteins of interest)Immobile phase- matrix that solvent moves through. Contains sites that proteins can bind to (or be trapped within) to slow down their movement. Greater interaction between the protein and the matrix, the longer it takes to move through the matrixchromatography to separate proteins based on some property of the amino acids/functionpart of purification process

several flavors:ion exchange gel filtrationaffinity

Chromatography: separating/isolating proteins

YouTube Video of IonExchange Chromatography332 phases:Mobile phase- solvent that is moving (includes proteins of interest)Immobile phase- matrix that solvent moves through. Contains sites that proteins can bind to (or be trapped within) to slow down their movement. Greater interaction between the protein and the matrix, the longer it takes to move through the matrixchromatography to separate proteins based on some property of the amino acids/functionpart of purification process

several flavors:ion exchange gel filtrationaffinity

Chromatography: separating/isolating proteins

342 phases:Mobile phase- solvent that is moving (includes proteins of interest)Immobile phase- matrix that solvent moves through. Contains sites that proteins can bind to (or be trapped within) to slow down their movement. Greater interaction between the protein and the matrix, the longer it takes to move through the matrixchromatography to separate proteins based on some property of the amino acids/functionpart of purification process

several flavors:ion exchange gel filtrationaffinityChromatography: separating/isolating proteins

352 phases:Mobile phase- solvent that is moving (includes proteins of interest)Immobile phase- matrix that solvent moves through. Contains sites that proteins can bind to (or be trapped within) to slow down their movement. Greater interaction between the protein and the matrix, the longer it takes to move through the matrix

SDS is an ionic detergentboiling, SDS, b-ME denatures protein3) imparts a negative charge4) disrupts disulfide bondsSDS-PAGE: separating proteinsPAGE= polyacrylamide gel electrophoresisproteins are separated by molecular weight in an electric field through a matrix of acrylamide. Matrix retards larger proteins more than smaller ones GO TO NEXT PPT Detergent and reducing BME removes all shape of protein (returns to primary structure) and gives all proteins similar negative charge, so the proteins are separated based upon size.36Called immunoglobulinsSynthesized by lymphocytes (white blood cells) in response to an antigen or foreign materialSpecific for a particular amino acid sequence on the antigenBinds tightly to antigenAnimals and Cell-lines are used to make antibodies for research and medicine

Antibodies are Proteins37Antibodies are Proteins

Image from: http://image.slidesharecdn.com/bt5proteinpart238 antibody-based techniques are widely used for studying proteins

Immunohistochemistry; immunofluorescence (Antibody staining) to look at protein localization in cells can also see if 2+ proteins co-localize to the same subcellular region of a cell (at low resolution).

Immunoblot (ie Western blot) use purified cells or pieces of tissue to make protein extracts. This uses a primary antibody, and a secondary antibody, which is usually coupled to an enzyme. Westerns show protein amount, size (molecular weight); generally more quantitative than antibody staining (can digitally measure expression).

Immunoprecipitation- purify proteins from cells, add antibody, secondary antibody linked to beads, incubate, spin down the beads and run out on a gel. This allows protein complexes to be pulled out. Can label proteins ahead of time or use a Western blot to identify proteins of interest. YY*Think about co-localization39

Antibodies in the LabImmunofluorescence40Ab against pectin in plant cell walls. FIX cells (crosslink proteins, break open membranes), add fluroescent antibodies and dyes if needed

antibody-based techniques are widely used for studying proteins

Immunohistochemistry; immunofluorescence (Antibody staining) to look at protein localization in cells can also see if 2+ proteins co-localize to the same subcellular region of a cell (at low resolution).

Immunoblot (ie Western blot) use purified cells or pieces of tissue to make protein extracts that are run on SDS-PAGE gels. This uses a primary antibody, and a secondary antibody, which is usually coupled to an enzyme. Westerns show protein amount, size (molecular weight)Immunoprecipitation- purify proteins from cells, add antibody, secondary antibody linked to beads, incubate, spin down the beads and run out on a gel. This allows protein complexes to be pulled out. Can label proteins ahead of time or use a Western blot to identify proteins of interest. Think about co-localization41

Antibodies in the LabWestern Blots42 antibody-based techniques are widely used for studying proteins

Immunohistochemistry; immunofluorescence (Antibody staining) to look at protein localization in cells can also see if 2+ proteins co-localize to the same subcellular region of a cell (at low resolution).

Immunoblot (ie Western blot) use purified cells or pieces of tissue to make protein extracts that are run on SDS-PAGE gels. This uses a primary antibody, and a secondary antibody, which is usually coupled to an enzyme. Westerns show protein amount, size (molecular weight)

Immunoprecipitation- purify proteins from cells, add antibody, secondary antibody linked to beads, incubate, wash all nonspecific interactions away, remove proteins from beads and run on a gel. This allows protein complexes to be pulled out. Think about co-localization43

Antibodies in the LabImmunoprecipitation44