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Ch4: How Proteins are Controlled

and Studied
BSC 300
Lecture 6
Tuesday, September 8

Diversity in enzyme funcJons

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Example of enzymaJc funcJon: Lysozyme

A class of enzyme found in egg white, tears, saliva, milk etc
Natural anJbioJcs: Sever pepJdoglycan chains in bacterial cell wall
(causing the cells to rupture)
Small, stable and easily harvested: First enzyme X-ray crystal structure
determined
Hydroly'c enzyme: Catalyzes nucleophilic aWack of glycosidic linkage by
water molecule. As discussed for ATP hydrolysis, the free energy of the
severed polysaccharide is less than the free energy of the intact chain.

Reviewing enzymaJc funcJons: Lysozyme

Recall acJvaJon energy and transiJon state
Such reacJons require the water molecule hit the bond with enough
force and at the correct angle impossible under normal condiJons
Lysozymes bind polysaccharides and distort conformaJon of a sugar in
the chain. This allows a chain reacJon of nucleophile subsJtuJon that
breaks the bond, lyse a water molecule and form more energeJcally
favorable products.

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Reviewing enzymaJc funcJons: Lysozyme

Induced t: Explains interacJon between
enzyme and substrate -- Enzymes and
substrate are exible, upon associaJon
conform to one anothers shape, leading to
opJmal interacJon and physical stress that
alters substrate conformaJon.
When associated the complex is referred to as
the enzyme-substrate complex
Key to enzyme acJvity is posiJon of specic
reacJve residues in the enzyme acJve site.
Polar or charged resides promote chemical
reacJons by serving as nucleophiles or
electrophiles.

Wikipedia

Lysosome catalyzed hydrolysis

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Enzymes can catalyze reacJon in several ways

Tightly bound small molecules add extra

funcJon to proteins
Proteins oben associate (electrostaJcally and covalently) with small
non-protein molecules which aide in protein funcJon.
Examples: reJnal and heme add funcJonality to the proteins
rhodopsin and hemoglobin, respecJvely.
May serve as a binding center: heme
binds O2 and CO2 in hemoglobin
May alter conformaJon of protein:
ReJnal is the light absorbing
component of rhodopsin. Absorbing
photons alters its conformaJon and
rhodopsin as a result.

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Coenzymes and cofactors

Add funcJonality to enzymes

Coenzymes: Organic molecules that are required by certain enzymes to
carry out catalysis. BioJn, for example associates with carboxylase
enzymes that transfer carboxyl groups (CO2) between substrates. Forms
transient covalent bond with carboxyl group, thereby acJng as an
ac$vated carrier.
Vitamins are precursors to
coenzymes

Saw this example in the last lecture in the

carboxylaJon of pyruvate.

Coenzymes and cofactors

Add funcJonality to enzymes

Cofactors: Inorganic molecules or ions that bind enzymes and are
essenJal for enzymaJc acJvity
Metal ions like Cu2+, Cu+, Zn2+, Fe3+, Fe2+ and Mg2+
Carboxypep$dase and other metalloproteases

hydrolyses pepJde chains (at specic residues) and
require zinc ions in their catalyJc sites to fold properly

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How proteins are controlled

Methods of regulaJng protein acJvity include:
Controlling when and where gene encoding protein is expressed
(transcripJonal control)
Controlling the subcellular localizaJon of a protein
Most rapid and general mechanism is altering protein conformaJon,
since protein funcJon is dictated by it adopJng an appropriate shape

There are many mechanisms of altering conformaJon in order to regulate
protein acJvity

CatalyJc acJvity of an enzyme is oben regulated

by binding of other molecules
AcJve site: Region of enzyme that binds substrate
CatalyJc site: Region of enzyme associate with acJve site that
catalyzes chemical reacJon, someJmes located within acJve site,
or brought into proximity following induced t
Regulatory site: Many enzymes contain binding sites for molecules
other than substrate. Binding of such molecules alters enzyme
conformaJon. Such binding can be either acJvaJng or repressing.

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CatalyJc acJvity of an enzyme is oben regulated

by binding of other molecules
Common regulatory mechanism for enzyme
acJvity is feedback inhibiJon: AccumulaJon
of specic product in an enzymaJc pathway
blocks acJvity of an enzyme in the pathway.
nega$ve regula$on.

Conversely, some metabolites (usually of
another pathway, or introduced
extracellularly) may bind enzyme regulatory
site and promote posi$ve regula$on.
Pathway metabolites create webs of
regulatory interacJons between pathways

Allosteric regulaJon
Allosteric regulaJon: Altered shape. Represents any binding of a
molecule to a site other than the acJve site - alters the conformaJon of
an enzyme.
Enzymes can adopt a range of conformaJons (2 or more). One
promotes substrate binding/catalysis, other conformaJons prevent
substrate binding.

NoncompeJJve inhibitors:
Bind to sites other than acJve
sites and inacJvate the enzyme.
Alter enzyme conformaJon
VMax of enzyme is not reached.
Cannot be overcome with high
substrate/inhibitor raJos.

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Allosteric regulaJon
Allosteric regulaJon can be posiJve as well as negaJve.

Reversible PhosphorylaJon
EnzymaJc transfer of phosphate group
between substrate molecules catalyzed by
kinase family of enzymes
Phosphate source = ATP, GTP, UTP or some
other acJvated substrate molecule
Phosphatases remove phosphate groups
from substrate
Two major kinases classes:
Serine/threonine
Tyrosine

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Roles of phosphorylaJon
Alters protein conformaJon
AcJvate/inacJvate enzyme acJvity
Signaling pathways
Charge molecules to drive chemical reacJons
Promote enzyme binding
Promote protein-protein interacJon (docking sites)
May target proteins for degradaJon by ubiquiJn/proteasome pathway
More than half of all proteins are phosphorylated to control cellular
processes
Most proteins phosphorylated at mulJple residues

The combinaJons of phosphorylaJon and other covalent modicaJons

signicantly alter one proteins acJvity producing range of acJviJes.

Other covalent modicaJons

UbiquiJn: A small regulatory protein. UbiquiJn ligases catalyze
transfer of this proteins to substrate proteins.
Roles:
Can alter protein subcellular localizaJon or acJvity
PolyubiquiJnaJon targets substrate proteins for destrucJon by
protein complexes called proteasomes.

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Other covalent modicaJons

AcetylaJon: Acetyltransferases catalyze covalent aWachment of
acetyl (CH3CO) groups to specic residues.
protein stability
localizaJon
acJvaJon

Many other forms of post-translaJonal

modicaJon

methylaJon
glycosylaJon
nitrosylaJon
lipidaJon
proteoloysis

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GTP-binding proteins
Aka G-proteins: CriJcal class of intracellular (inside the cell) signaling
molecules relay informaJon from signal receptor at the cell surface to
other proteins within the cell.
AcJvity is regulated by binding to and hydrolyzing GTP (guanine
nucleoJde diphosphate). Bound to GTP the protein is in an on
conformaJon, once GTP has been hydrolyzed to GDP the G-protein is
inacJve (details in CH 16).

Motor proteins
Intracellular transport and other acJviJes like
chromosome segregaJon and muscle contracJon
are driven by molecular motor proteins (ie
dynein, kinesin, myosin).
The motors are ATPases, (hydrolyze ATP) whose
change in conformaJon is driven by binding to
and hydrolyzing ATP.

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Protein machines
Proteins can self-assemble into nanoscale machines that exhibit
sophisJcated funcJonal properJes and can perform work using chemical
energy, typically obtained from the hydrolysis of ATP or GTP

Ex: DNA/RNA polymerases, ribosomes, ATPsynthase, proteasomes,

chaperones, pyruvate dehydrogenase

How proteins are studied

Understanding their funcJons requires detailed understanding of:

Structure
Biochemistry
InteracJons with other proteins
Cellular and subcellular localizaJon
Expression levels
Post-translaJonal modicaJons
etc, etc

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ExtracJon from Jssue/cells

Various techniques can
be used to homogenize
Jssue/cell culture. This
disrupts cell membrane
and frees cell contents
without disrupJng
membrane of subcellular
organelles.

FracJonaJng the homogenate

High speed centrifugaJon separates cytosolic components. A variety of
techniques can be employed to separate components based on mass, size,
buoyancy, solubility (see Panel 4-3).
This can be used to fracJonate specic organelles, just the cytoplasm or
membrane
Successive rounds of
centrifugaJon can be used
to fracJonate organelle

proteions

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Purifying proteins from soluJon

ResulJng slurry is a mixture of many dierent proteins
Other techniques allow this mix to be further rened: Chromatography

Examples of Chromatography
Ion exchange separates proteins based on
electric charge. Gel ltraJon by size
dicult to purify a single protein of interest.
Anity chromatography: Allows for selecJve
protein puricaJon. Column matrix is
covalently aWached to a bait molecule.
Bait may be a protein for which you wish to
idenJfy interacJng proteins.
Bait may be an anJbody that specically
reacts with your protein of interest.

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Examples of Chromatography
Protein slurry passes through anity column
and only specic interacJons occur all
other proteins pass through column.
Bound protein is then recovered (eluted) by
passing a soluJon of high salt or altered pH
through the column.

Protein separaJon by electrophoresis

Gels are usually denaturing,
meaning they will disrupt
electrostaJc interacJons and
reduce disulde bonds.
Proteins will migrate through
an acrylamide gel, toward the
posiJve anode based on
weight and net charge.

Separated proteins can be
extracted as individual bands
from gel.

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2-D gel electrophoresis

Powerful method for separaJng all
proteins within a cell culture or
Jssue.

1. Isoelectric focusing: Non-

denatured proteins separated
based on total charge

< ---------- size ----------- >

Proteins separated in two ways:

basic < -----------pH ----------- > acidic

2. Molecular weight: Then

transferred to a denaturing gel and
separated based on mol. weight.

Determining protein sequence

Sequence of an isolated unknown
protein can be determined using mass
spectrometry
Protein is digested into smaller
fragments by a protease and these
fragments ionized by a laser blast.
Fragments then separated in an electric
eld based on mass to charge raJo
Protein sequence can be predicted base
on genomic sequences are searched for
matches to these fragment raJos.

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Determining protein structure

X-ray crystallography and
nuclear magneJc resonance
Homework reading. Answers
due 9/11. Will not be exam I
material.

Vertebrate anJbodies: Example of binding

diversity
Each chain composed of repeated units of -sheets whose
structures are held in place by disulde bonds
At the Jp of each arm is the anJgen binding site which recognizes
a specic ligand, the anJgen (viral parJcle, bacterial protein, or any
foreign molecule that the host has been exposed to).

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ProducJon and uses of anJbodies

Important experimental and clinical uses:
Experimental: AnJbodies against
specic epitopes (parts of molecules)
can be used to tag those molecules
within xed cells, Jssues or extract.
The anJbody can then be bound by a
second anJbody that is linked to a
uorescent, radioacJve or
colorometric molecule. As a result,
researchers can visualize where within
the cell/Jssue the molecule of interest
is, or whether the molecule is in the
sample at all.

ProducJon and uses of anJbodies

Important experimental and clinical uses:

Experimental:
Can be used to bind and purify proteins from cells for further
study.

AnJbodies may also be used to treat a living Jssue or cell culture.

Binding the anJgen prevents the normal funcJon of the molecule,
so anJbody treatment results in a loss of funcJon phenotype.

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ProducJon and uses of anJbodies

Important experimental and clinical uses:

Clinical: Monoclonal an$body therapy: AnJbodies are becoming
powerful tools to baWle specic diseases, especially cancer.

May bind and inhibit a rogue protein central to cancer
metastasis
May be conjugated to an toxin for direct delivery to cancer
cells
May bind cancer cells and sJmulate the immune response
May block molecules that inhibit the immune system (oben
produced by the cancer cells)
May block molecules that promote cell division in cancer

Making anJbodies
Two types:
Polyclonal anJbodies: A collecJon of anJbodies puried
from the blood serum of a laboratory animal. It contains
many unique species of anJbody that bind the same
anJgen, but may bind with dierent aniJes and bind the
anJgen at dierent locaJons.
Monoclonal anJbodies: Produced from a clonal populaJon
of B cells. Monoclonal anJbodies against an anJgen are
idenJcal to one another, bind with the same anity and
to the exact same epitope.

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Making polyclonal anJbodies

Purify the anJgen, and inject
laboratory animal
Polyclonal: Booster shots sJmulate B
cells to produce more of the same
anJbodies
Exsanguinate the animal: Draw all of
its blood. Centrifuge blood to collect
serum. This contains a high
concentraJon of dierent anJbodies
against the anJgen.
Can be further puried by using the
anJgen to isolate reacJve anJbodies

Making polyclonal anJbodies

Following anJgen injecJon, the
animal is sacriced and individual
B cells from the animals spleen
are fused with cancer cells.
The resulJng hybridomas can be
cultured indenitely, even frozen.
Each cultured cell line secretes
large quanJJes of a single
anJbody. These can be tested for
reacJvity and individual cell lines
selected for use.

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