Download Protein Structure & Function

Chapter 4
Protein Structure
and Function
Proteins


Make up about 15% of the cell
Have many functions in the cell









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

Folding@home



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:


Fold@home web site: http://folding.stanford.edu/
Article on Folding@home:
http://www.sciencedaily.com/releases/2002/10/02102
2070813.htm
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.
Levels of Organization

Primary structure


Amino acid sequence of the protein
Secondary structure

H bonds in the peptide chain backbone
 -helix

Tertiary structure


and -sheets
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
 globin
subunits
 2  globin
subunits
2
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

– fiber with a glycine every third amino
acid in the protein
 Elastin – unstructured fibers that gives tissue an
elastic characteristic
 Collagen
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


When a prosthetic group is required by an
enzyme it is called a co-enzyme


Hemoglobin requires heme (iron containing
compound) to carry the O2
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, transmembrane signaling
etc.
Usually driven by ATP
or GTP hydrolysis
See video clip on CD in
book