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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