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
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
• Quaternary 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
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
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 1 st 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 H 2O 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 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
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 PO 4 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