Transcript Protein Structure & Function

Protein Structure and Function
Proteins
• 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
Hydrophilic
Figure 3-2 Molecular Biology of the Cell (© Garland Science 2008)
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
Figure 3-4 Molecular Biology of the Cell (© Garland Science 2008)
Globular Proteins
• The side chains will help determine the conformation in
an aqueous solution
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)
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
Figure 3-6a Molecular Biology of the Cell (© Garland Science 2008)
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
-helix
Figure 3-7a,b,c Molecular Biology of the Cell (© Garland Science 2008)
-sheet
Figure 3-7d,e,f Molecular Biology of the Cell (© Garland Science 2008)
 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)
Figure 3-8 Molecular Biology of the Cell (© Garland Science 2008)
 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
Figure 3-9 Molecular Biology of the Cell (© Garland Science 2008)
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
Figure 3-12 Molecular Biology of the Cell (© Garland Science 2008)
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
Figure 3-36 Molecular Biology of the Cell (© Garland Science 2008)
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
Figure 3-41 Molecular Biology of the Cell (© Garland Science 2008)
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
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
Figure 3-50a Molecular Biology of the Cell (© Garland Science 2008)
Features of Enzyme Catalysis
Figure 3-52 Molecular Biology of the Cell (© Garland Science 2008)
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 non-covalently
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
Figure 3-71 Molecular Biology of the Cell (© Garland Science 2008)
Molecular Switches
Figure 3-75 Molecular Biology of the Cell (© Garland Science 2008)
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
Figure 3-76 Molecular Biology of the Cell (© Garland Science 2008)
Protein Machines
• Complexes of 10 or more proteins that work together such
as DNA replication, RNA or protein synthesis, transmembrane signaling etc.
• Usually driven by ATP or GTP hydrolysis
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