Lecture 4 learning goals

Describe the key factors that determine

protein structure
Explain why the structure of proteins is
important
Describe mechanisms that regulate
how proteins are controlled
Techniques used to study proteins
How 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?

Online Protein Introduction

Video

Clicker Question
Review!!!

Protein Structure

Protein Structure

Tertiary
Structure

Secondary
Structure

Secondary
Protein
Structure
largely a result of interactions between amino acid backbones in the
polypeptide chain

alpha helix

beta-pleated sheet

not all regions of proteins are organized into these structures. There are
regions within proteins that are disordered or flexible.

Secondary
Protein
Structure
largely a result of interactions between amino acid backbones in the
polypeptide chain

alpha helix

beta-pleated sheet

not all regions of proteins are organized into these structures. There are
regions within proteins that are disordered or flexible.

Many proteins are embedded in

membrane

The method of protein integration

into the membrane is not
spontaneous.
We will learn more about this in

Bonds that Maintain Protein

Structures
non-covalent bonds

s
o nd
b
t
len
a
v
o
c

side chains

Hydrophobic Forces Drive Protein

Folding

amino acids that are commonly modified:

Post-translational Modifications
Disulfide bonds
between cysteines

Cytosol is a
highly
reducing
environment

Many
extracellular
proteins are
held
together by
disulfide
bonds

Phosphorylation on
serine, threonine, or
tyrosine

amino acids that are commonly modified:

Post-translational Modifications
Glycosylation on asparagine,
serine, and threonine

N-linked

O-linked

Ubiquitination on lysine

Some proteins have multiple

domains

Src protein has 4 distinct domains

Domain: different regions of the

polypeptide chain fold
independently

Fibronectin (ECM)
has 4 successive
domains of the

Proteins can share domain structure

may be well-conserved
regions appearing in
many
proteins/organisms

C
which protein might this be?

Different Visual
Representations of Protein
Structure

4 Models:
Backbone (chain)
Ribbon model
Wire model
Space filling model

Quaternary Protein Structure =

Multi-subunit proteins

dimers,
trimers,
tetramers,
etc..
Usually linked
by noncovalent
bonds and/or
hydrophobic
surfaces
Can be made
up of heteroor homoassociations

Protein Folding can create Binding

Sites

protein case study:

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

hemoglobin schematic from wikipedia commons

when O2 binds to
hemoglobin, it changes
the tertiary and
quaternary structure of
the complex

protein case study:

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

hemoglobin schematic from wikipedia commons

when O2 binds to
hemoglobin, it changes
the tertiary and
quaternary structure of
the complex

How Proteins are Controlled

Protein levels (gene expression, protein
production, protein degradation)
Confining Proteins to Subcellular compartments
Alter Protein Conformation (and thus its
function)
Most proteins are allosteric
Have two (or more) slightly different
conformations
Protein activity can be regulated by
shifting between these conformations
Binding of a regulatory protein/molecule
Covalent modifications on Proteins
Phosphorylation
Acetylation
Ubiquitination

How Proteins are Controlled

Protein levels (gene expression, protein
production, protein degradation)
Confining Proteins to Subcellular compartments
Alter Protein Conformation (and thus its
function)
Most proteins are allosteric
Have two (or more) slightly different
conformations
Protein activity can be regulated by
shifting between these conformations
Binding of a regulatory protein/molecule
Covalent modifications on Proteins
Phosphorylation
Acetylation
Ubiquitination

How Proteins are Controlled

Protein levels (gene expression, protein
production, protein degradation)
Confining Proteins to Subcellular compartments
Alter Protein Conformation (and thus its
function)
Most proteins are allosteric
Have two (or more) slightly different
conformations
Protein activity can be regulated by
shifting between these conformations
1) Binding of a regulatory
protein/molecule
2) Covalent modifications on Proteins
Phosphorylation
Acetylation

How Proteins are Controlled

Protein levels (gene expression, protein
production, protein degradation)
Confining Proteins to Subcellular compartments
Alter Protein Conformation (and thus its
function)
Most proteins are allosteric
Have two (or more) slightly different
conformations
Protein activity can be regulated by
shifting between these conformations
1) Binding of a regulatory protein/molecule
2) Covalent modifications on Proteins
Phosphorylation
Acetylation
Ubiquitination

How Proteins are Controlled

Protein levels (gene expression, protein
production, protein degradation)
Confining Proteins to Subcellular compartments
Alter Protein Conformation (and thus its
function)
Most proteins are allosteric
Have two (or more) slightly different
conformations
Protein activity can be regulated by
shifting between these conformations
Binding of a regulatory protein/molecule
Covalent modifications on Proteins
Phosphorylation
Acetylation
Image
from: http://biomatics.org/index.php/Acetylation
Ubiquitination

How Proteins are Controlled

Protein levels (gene expression, protein
production, protein degradation)
Confining Proteins to Subcellular compartments
Alter Protein Conformation (and thus its
function)
Most proteins are allosteric
Have two (or more) slightly different
conformations
Protein activity can be regulated by
shifting between these conformations
Binding of a regulatory protein/molecule
Covalent modifications on Proteins
Phosphorylation
Acetylation
Image
by: Jennifer Kowalski (Butler University)
Ubiquitination

Composition and Structure can be determined

for
purified proteins
mass spectrometry
Determines protein sequence or
identity
Proteins often first digested with
trypsin
Measure mass and determine
chemical species within
fragments

Composition and Structure can be determined

for
purified proteins

x-ray crystallography
Uses crystals of purified
proteins and determines (up
to) tertiary structure
Bombard crystal with X-rays
and then collect diffraction
information

Composition and Structure can be determined

for
purified proteins
nuclear magnetic resonance (NMR)
spectroscopy
Uses small-sized purified protein and determines (up
to) tertiary structure
Incubate protein solution in magnetic field and
collect information about positioning of hydrogen
nuclei

Protein
solution

NMR spectrum
NMR Magnet

Composition and Structure can be determined

for
purified proteins

So how do we purify a protein?

1. Break open cells
2. Separate proteins from other
cellular components (nucleic
acids, lipids)
3. Separate your protein of
interest away from all other
proteins within the cell

Homogenization: breaking open cells

Rupturing the plasma membrane to release cell
contents

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Centrifugation: separating components within

homogenant

Differential Centrifugation

Chromatography: separating/isolating proteins

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

Chromatography: separating/isolating proteins

chromatography to
separate proteins based on
some property of the
amino acids/function
part of purification
process
several flavors:
ion exchange
gel filtration
(affinity)

Chromatography: separating/isolating proteins

chromatography to
separate proteins based on
some property of the
amino acids/function
part of purification
process
several flavors:
ion exchange
gel filtration
affinity

YouTube Video of IonExchange Chromatography

Chromatography: separating/isolating proteins

chromatography to
separate proteins based on
some property of the
amino acids/function
part of purification
process
several flavors:
ion exchange
gel filtration
affinity

Chromatography: separating/isolating proteins

chromatography to
separate proteins based on
some property of the
amino acids/function
part of purification
process
several flavors:
ion exchange
gel filtration
affinity

SDS-PAGE: separating proteins

PAGE= polyacrylamide gel electrophoresis
1)
2)
3)
4)

proteins are separated by molecular

weight in an electric field through a matrix
of acrylamide. Matrix retards larger
proteins more than smaller ones

SDS is an ionic detergent

boiling, SDS, -ME denatures prote
imparts a negative charge
disrupts disulfide bonds

Antibodies are Proteins

Called immunoglobulins
Synthesized by lymphocytes (white
blood cells) in response to an antigen
or foreign material
Specific for a particular amino acid
sequence on the antigen
Binds tightly to antigen

Animals and Cell-lines are used to make

antibodies for research and medicine

Antibodies are Proteins

Image from: http://image.slidesharecdn.com/bt5proteinpart

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

Y*

Antibodies in the Lab

Immunofluorescence

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.

Antibodies in the Lab

Western Blots

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.

Antibodies in the Lab

Immunoprecipitation