Transcript Protein Structure & Function - Lectures For UG-5

Protein Structure
and Function
Proteins
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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
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Proteins are made of 20 amino acids linked by peptide
bonds
Polypeptide backbone is the repeating sequence of the NC-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
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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
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The side chains will help determine the
conformation in an aqueous solution
Hydrogen Bonds in Proteins
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H-bonds form between 1) atoms involved in the
peptide bond; 2) peptide bond atoms and R groups; 3)
R groups
Protein Folding
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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
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Can be down with heat, pH or chemical compounds
In the chemical compound, can remove and have the
protein renature or refold
Folding@home
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The Stanford Folding@home research goal is to
understand protein folding, misfolding, and related
diseases.
Calculations to create models requires a
supercomputer OR many smaller computers
(distributed computing).
You can participate by visiting:
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Fold@home web site: http://folding.stanford.edu/
Article on Folding@home:
http://www.sciencedaily.com/releases/2002/10/02102207081
3.htm
Refolding
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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
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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
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Core of many proteins is
the  sheet
Form rigid structures with
the H-bond
Can be of 2 types
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Anti-parallel – run in an
opposite direction of its
neighbor (A)
Parallel – run in the same
direction with longer looping
sections between them (B)
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 Helix
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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
Levels of Organization
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Primary structure
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Amino acid sequence of the protein
Secondary structure
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H bonds in the peptide chain backbone
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Tertiary structure
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-helix and -sheets
Non-covalent interactions between the R groups within the
protein
Quanternary structure
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Interaction between 2 polypeptide chains
Protein Structure
Domains
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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
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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
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Have similarities in amino acid sequence and 3-D
structure
Have similar functions such as breakdown proteins
but do it differently
Proteins – Multiple Peptides
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Non-covalent bonds can form interactions between
individual polypeptide chains
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Binding site – where proteins interact with one another
Subunit – each polypeptide chain of large protein
Dimer – protein made of 2 subunits
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Can be same subunit or different subunits
Single Subunit Proteins
Different Subunit Proteins
 Hemoglobin
 globin
subunits
 2  globin
subunits
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Protein Assemblies
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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
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Globular Proteins – most of what we have dealt with
so far
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Compact shape like a ball with irregular surfaces
Enzymes are globular
Fibrous Proteins – usually span a long distance in the
cell
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3-D structure is usually long and rod shaped
Important Fibrous Proteins
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Intermediate filaments of the cytoskeleton
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Structural scaffold inside the cell
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Keratin in hair, horns and nails
Extracellular matrix
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Bind cells together to make tissues
Secreted from cells and assemble in long fibers
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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
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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
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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
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Consists of a cavity formed by a specific arrangement of
amino acids
Ligand Binding
Formation of Binding Site
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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
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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
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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
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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
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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 noncovalent bonds that hold the 2 together
Enzymes as Catalysts
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Enzymes are proteins that bind to their ligand as the
1st step in a process
An enzyme’s ligand is called a substrate
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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
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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
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Non-covalent bonds hold the polysaccharide in the
active site until the reaction occurs
Features of Enzyme Catalysis
Enzyme Performance
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E + S  ES  EP  E + P
Step 1 – binding of the substrate
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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
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State of substrate prior to becoming product
Enzymes lowers the energy of transition state and
therefore accelerates the reaction
Reaction Rates
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KM – [S] that allows rxn to proceed at ½ it
maximum rate
Prosthetic Groups
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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
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When a prosthetic group is required by an
enzyme it is called a co-enzyme
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Hemoglobin requires heme (iron containing compound)
to carry the O2
Usually a metal or vitamin
These groups may be covalently or non-covalently
linked to the protein
Regulation of Enzymes
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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
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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
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 ADP levels cause the
activation of the glycolysis
pathway to make more ATP
Allostery
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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
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Method of regulation is also used in other
proteins besides enzymes
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Receptors, structural and motor proteins
Allosteric Regulation
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Enzyme is only partially active with sugar only but much
more active with sugar and ADP present
Phosphorylation
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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
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Kinases capable of
putting the PO4 on 3
different amino acid
residues
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Have a –OH group on R
group
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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)
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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
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Motor Proteins
Proteins can move in the cell,
say up and down a DNA strand
but with very little uniformity
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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
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The motor proteins that move
things along the actin filaments
or myosin
Protein Machines
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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
See video clip on CD in
book