Transcript Chapter 4

Chapter 4
Protein Structure
and Function
Proteins
• Make up about 15% of the cell
• Have many functions in the cell
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Enzymes
Structural
Transport
Motor
Storage
Signaling
Receptors
Gene regulation
Special functions
Shape = Amino Acid Sequence
• Proteins are made of 20 amino acids
linked by peptide bonds
• Polypeptide backbone is the repeating
sequence of the N-C-C-N-C-C… in the
peptide bond
• The side chain or R group is not part
of the backbone or the peptide bond
Polypeptide
Backbone
Amino Acids
NOTE: You need to know this table
Hydrophilic
Hydrophobic
Protein Folding
• The peptide bond allows for rotation
around it and therefore the protein
can fold and orient the R groups in
favorable positions
• Weak non-covalent interactions will
hold the protein in its functional
shape – these are weak and will take
many to hold the shape
Non-covalent Bonds in Proteins
Globular Proteins
• The side chains will help determine the
conformation in an aqueous solution
Hydrogen Bonds in Proteins
• H-bonds form between 1) atoms involved in the
peptide bond; 2) peptide bond atoms and R
groups; 3) R groups
Protein Folding
• Proteins shape is determined by the
sequence of the amino acids
• The final shape is called the
conformation and has the lowest free
energy possible
• Denaturation is the process of
unfolding the protein
– Can be down with heat, pH or chemical
compounds
– In the chemical compound, can remove
and have the protein renature or refold
Refolding
• Molecular chaperones are small proteins
that help guide the folding and can help
keep the new protein from associating with
the wrong partner
Protein Folding
• 2 regular folding
patterns have been
identified – formed
between the bonds of
the peptide backbone
• -helix – protein turns
like a spiral – fibrous
proteins (hair, nails,
horns)
• -sheet – protein folds
back on itself as in a
ribbon –globular protein
 Sheets
• Core of many proteins
is the  sheet
• Form rigid structures
with the H-bond
• Can be of 2 types
– Anti-parallel – run in an
opposite direction of its
neighbor (A)
– Parallel – run in the
same direction with
longer looping sections
between them (B)
 Helix
• Formed by a H-bond
between every 4th
peptide bond – C=O to
N-H
• Usually in proteins that
span a membrane
• The  helix can either
coil to the right or the
left
• Can also coil around each
other – coiled-coil shape
– a framework for
structural proteins such
as nails and skin
CD from Text
• The CD that is included on your
textbook back cover has some video
clips that will show the  helix and 
sheets as well as other things in this
chapter. You will want to look at
them. If you have problems, we will
look at them during lab.
Levels of Organization
• Primary structure
– Amino acid sequence of the protein
• Secondary structure
– H bonds in the peptide chain backbone
• -helix and -sheets
• Tertiary structure
– Non-covalent interactions between the R
groups within the protein
• Quanternary structure
– Interaction between 2 polypeptide chains
Protein Structure
Domains
• A domain is a basic structural unit of
a protein structure – distinct from
those that make up the
conformations
• Part of protein that can fold into a
stable structure independently
• Different domains can impart
different functions to proteins
• Proteins can have one to many
domains depending on protein size
Domains
Useful Proteins
• There are thousands and thousands of
different combinations of amino acids that
can make up proteins and that would
increase if each one had multiple shapes
• Proteins usually have only one useful
conformation because otherwise it would
not be efficient use of the energy available
to the system
• Natural selection has eliminated proteins
that do not perform a specific function in
the cell
Protein
Families
• Have similarities in amino acid sequence
and 3-D structure
• Have similar functions such as breakdown
proteins but do it differently
Proteins – Multiple Peptides
• Non-covalent bonds can form
interactions between individual
polypeptide chains
– Binding site – where proteins interact
with one another
– Subunit – each polypeptide chain of large
protein
– Dimer – protein made of 2 subunits
• Can be same subunit or different subunits
Single Subunit Proteins
Different Subunit Proteins
• Hemoglobin
– 2  globin
subunits
– 2  globin
subunits
Protein Assemblies
• Proteins can form
very large assemblies
• Can form long chains
if the protein has 2
binding sites – link
together as a helix or
a ring
• Actin fibers in
muscles and
cytoskeleton – is made
from thousands of
actin molecules as a
helical fiber
Types of Proteins
• Globular Proteins – most of what we
have dealt with so far
– Compact shape like a ball with irregular
surfaces
– Enzymes are globular
• Fibrous Proteins – usually span a long
distance in the cell
– 3-D structure is usually long and rod
shaped
Important Fibrous Proteins
• Intermediate filaments of the
cytoskeleton
– Structural scaffold inside the cell
• Keratin in hair, horns and nails
• Extracellular matrix
– Bind cells together to make tissues
– Secreted from cells and assemble in long
fibers
• Collagen – fiber with a glycine every third
amino acid in the protein
• Elastin – unstructured fibers that gives tissue
an elastic characteristic
Collagen and Elastin
Stabilizing Cross-Links
• Cross linkages can be between 2 parts of a
protein or between 2 subunits
• Disulfide bonds (S-S) form between adjacent SH groups on the amino acid cysteine
Proteins at Work
• The conformation of a protein gives it a
unique function
• To work proteins must interact with other
molecules, usually 1 or a few molecules
from the thousands to 1 protein
• Ligand – the molecule that a protein can
bind
• Binding site – part of the protein that
interacts with the ligand
– Consists of a cavity formed by a specific
arrangement of amino acids
Ligand Binding
Formation of Binding Site
• The binding site forms when amino acids from
within the protein come together in the folding
• The remaining sequences may play a role in
regulating the protein’s activity
Antibody Family
• A family of proteins that can be
created to bind to almost any
molecule
• Antibodies (immunoglobulins) are
made in response to a foreign
molecule ie. bacteria, virus, pollen…
called the antigen
• Bind together tightly and therefore
inactivates the antigen or marks it
for destruction
Antibodies
• Y-shaped molecules with 2 binding
sites at the upper ends of the Y
• The loops of polypeptides on the end
of the binding site are what imparts
the recognition of the antigen
• Changes in the sequence of the loops
make the antibody recognize
different antigens - specificity
Antibodies
Binding Strength
• Can be measured directly
• Antibodies and antigens are mixing around
in a solution, eventually they will bump into
each other in a way that the antigen sticks
to the antibody, eventually they will
separate due to the motion in the
molecules
• This process continues until the equilibrium
is reached – number sticking is constant
and number leaving is constant
• This can be determined for any protein and
its ligand
Equilibrium
Constant
• Concentration of antigen, antibody and
antigen/antibody complex at equilibrium can be
measured – equilibrium constant (K)
• Larger the K the tighter the binding or the more
non-covalent bonds that hold the 2 together
Enzymes as Catalysts
• Enzymes are proteins that bind to their
ligand as the 1st step in a process
• An enzyme’s ligand is called a substrate
– May be 1 or more molecules
• Output of the reaction is called the product
• Enzymes can repeat these steps many times
and rapidly, called catalysts
• Many different kinds – see table 5-2, p 168
Enzymes at Work
• Lysozyme is an important enzyme that
protects us from bacteria by making holes
in the bacterial cell wall and causing it to
break
• Lysozyme adds H2O to the glycosidic bond
in the cell wall
• Lysozyme holds the polysaccharide in a
position that allows the H2O to break the
bond – this is the transition state – state
between substrate and product
• Active site is a special binding site in
enzymes where the chemical reaction takes
place
Lysozyme
• Non-covalent bonds hold the polysaccharide in
the active site until the reaction occurs
Features of Enzyme Catalysis
Enzyme Performance
E + S  ES  EP  E + P
• Step 1 – binding of the substrate
– Limiting step depending on [S] and/or [E]
– Vmax – maximum rate of the reaction
– Turnover number determines how fast the
substrate can be processed = rate of rxn  [E]
• Step 2 – stabilize the transition state
– State of substrate prior to becoming product
– Enzymes lowers the energy of transition state
and therefore accelerates the reaction
Reaction Rates
• KM – [S] that allows rxn to proceed at ½ it
maximum rate
Prosthetic Groups
• Occasionally the sequence of the protein is
not enough for the function of the protein
• Some proteins require a non-protein
molecule to enhance the performance of
the protein
– Hemoglobin requires heme (iron containing
compound) to carry the O2
• When a prosthetic group is required by an
enzyme it is called a co-enzyme
– Usually a metal or vitamin
• These groups may be covalently or noncovalently linked to the protein
Regulation of Enzymes
• Regulation of enzymatic
pathways prevent the
deletion of substrate
• Regulation happens at
the level of the enzyme
in a pathway
• Feedback inhibition is
when the end product
regulates the enzyme
early in the pathway
Feedback Regulation
• Negative feedback –
pathway is inhibited by
accumulation of final
product
• Positive feedback – a
regulatory molecule
stimulates the activity
of the enzyme, usually
between 2 pathways
–  ADP levels cause the
activation of the
glycolysis pathway to
make more ATP
Allostery
• Conformational coupling of 2 widely
separated binding sites must be
responsible for regulation – active site
recognizes substrate and 2nd site
recognizes the regulatory molecule
• Protein regulated this way undergoes
allosteric transition or a conformational
change
• Protein regulated in this manner is an
allosteric protein
Allosteric Regulation
• Method of regulation is also used in other
proteins besides enzymes
– Receptors, structural and motor proteins
Allosteric Regulation
• Enzyme is only partially active with sugar only
but much more active with sugar and ADP
present
Phosphorylation
• Some proteins are regulated by the
addition of a PO4 group that allows
for the attraction of + charged side
chains causing a conformation change
• Reversible protein phosphorylations
regulate many eukaryotic cell
functions turning things on and off
• Protein kinases add the PO4 and
protein phosphatase remove them
Phosphorylation/Dephosphorylation
• Kinases capable of
putting the PO4 on 3
different amino acid
residues
– Have a –OH group on
R group
• Serine
• Threonine
• Tyrosine
• Phosphatases that
remove the PO4 may
be specific for 1 or 2
reactions or many be
non-specific
GTP-Binding Proteins (GTPases)
• GTP does not release its
PO4 group but rather the
guanine part binds tightly
to the protein and the
protein is active
• Hydrolysis of the GTP to
GDP (by the protein itself)
and now the protein is
inactive
• Also a family of proteins
usually involved in cell
signaling switching proteins
on and off
Molecular Switches
Motor Proteins
• Proteins can move in the
cell, say up and down a
DNA strand but with very
little uniformity
– Adding ligands to change
the conformation is not
enough to regulate this
process
• The hydrolysis of ATP can
direct the the movement
as well as make it
unidirectional
– The motor proteins that
move things along the
actin filaments or myosin
Protein Machines
• Complexes of 10 or
more proteins that
work together such as
DNA replication, RNA
or protein synthesis,
trans-membrane
signaling etc.
• Usually driven by ATP
or GTP hydrolysis
• See video clip on CD in
book