Biochemistry- Protein Structure

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Proteins
– linear polymers of amino acids
– peptide bond formation enables protein formation
Size of a protein
50 to >30 000 amino acids
Protein Formation:
1. Condensation reaction
2. Covalent bond (shares electrons)
3. No rotation around the bond
4. Atoms are co-planar
5. Restricts the possible folding patterns of the chain
Polypeptides
– small numbers of joined residues (amino acids)
– di-peptide, tri-peptide, etc (polypeptide)
– even proteins can be referred to as polypeptides
Polypeptide Backbone
– repeating part of polypeptide
-N-C-N-C-C-N-C-C
– at the 5′ end
Side Chains
project from backbone
Levels of protein structure
– proteins can fold into many different shapes
– certain structural elements are found in many proteins
– general “rules” guiding how proteins obtain final form
Primary Structure
– the sequence of residues (amino acids)
– polarity: N- and C- terminal ends (polar because have a + charge at one end and a – charge at the other end)
– the free amino terminal group is bonded to something else, therefore it is less flexible
– proline breaks any structure because it is an amino acid (note very flexible)
How do unsaturated proteins function?
– an UNSATURATED protein is much MORE flexible therefore those proteins can be much more complicated than was thought
– unsaturated proteins are like elastic bands
Secondary Structure
– local folding pattern of the backbone
– stabilized by hydrogen bonds (between backbone N-H and C=O groups)
2 major types of secondary structure:
1. alpha helix
2. beta sheet
Alpha Helix (secondary structure)
– coil spring arrangement
– 3.6 residues per turn
– forms a rod or cylindrical shape
– 0.54nm per turn
– side chains project outward
– no interior space
– H-bond stabilization
– N-H bonded to C=O later on in chain
– does not usually include proline residues
– proline causes kink
– has a tightly packed hydrophobic core
– stabilized by hydrogen bonds (not by hydrophobic bond/ionic bonds)
– example of secondary structure
Aromatase
– unstructured regions
– have a bunch of alpha helices
– most proteins have more than 1 secondary structure, but aromatase only has 1
Beta Sheet (secondary structure)
– hydrogen bond occurs between the backbone of 2 or more polypeptide chains arranged adjacently and in parallel
– side chains project up and down
– beta sheets could be parallel or anti-parallel; could happen between 2 adjacent peptides or between the same peptide
– can occur in varying amounts in soluble proteins
– is NOT a flexible structure
– is stabilized mainly by intrachain hydrogen bonding of the polypeptide backbone (therefore found in many different proteins)
– is found in fibroin
Summary of Secondary Structure
– silk fibroin consists mostly of beta sheets
– most proteins have both alpha helices and beta sheets, plus turns and other less regular structure
– primary sequence determines type of structure formed
Tertiary Structure
– concerns the 3D arrangement of the polypeptide chain
– interactions between residues distant in sequence
– gives overall shape of protein
– stabilized by multiple weak bonds and a very important covalent bond–> hydrophobic interactions between R groups, hydrogen bonding between R groups, electrostatic interactions between R groups, disulfide bond formation (NOT by amide bond formation)
– can be disrupted by: exposure to denaturing agents such as urea, high salt concentrations, organic solvents, hydrogen bonding disrupters (urea), proteases (NOT disrupted by purification by gel filtration)
– does NOT involve interactions between different polypeptide chains (consists of ONE polypeptide chain only)
– charged side chains are mainly on the exterior
4 weak bonds that Tertiary structure is stabilized by:
1. Hydrophobic interactions
2. Hydrogen bonds
3. Van der Waals forces
4. Ionic bonds (electrostatic interactions)
Covalent bond that Tertiary structure is stabilized by:
Disulphide bond
Disulphide bond
– bond between cysteine residues in distant parts of the protein
– only forms in oxidizing environments
– commonly formed in extracellular proteins
– are NOT formed by the cross-linking of methionine residues
– are NOT formed mainly in proteins that are retained in the cytosol
– can NOT be broken by oxidation through agents such as mercaptoethanol
– methionine has an S bond, but doesn’t create disulphide bonds
– stabilize, a protein, but does not change the overall final conformation
(see note for image – interchain and intrachain disulfide bonds)
Hydrophobic Interactions
– hydrophobic side chains “hidden” from water
– cluster together; exclude water from interior
– charged side chains are mainly exterior
– hydrophobic core contains non-polar side chains
– hydrogen bonds can be formed to the polar side chains on the outside of the molecule
Hydrogen Bonds
– covalent bond (physically connected)
– ex. H-N, H-O
– formed when a hydrogen atom is “sandwiched” between 2 electron-attracting atoms (usually O or N)
– can form in many different places (between atoms of 2 peptide bonds, between atoms of a peptide bond and an amino acid side chain, between 2 amino acid side chains)- between 2 adjacent water molecules
– strongest when the 3 atoms are in a straight line
– 1/20th the strength of a covalent bond (more energy required to break a peptide bond than hydrogen bond)
– stabilizes hydrogen bonds
– are susceptible to breakage by urea
– hold water molecules together
– important in maintaining secondary structure
– an interchain hydrogen bond is as strong as an intrachain hydrogen bond
Van der Waals forces
– sum of the attractive or repulsive forces between molecules (or between parts of the same molecule) other than those due to covalent bonds, hydrogen bonds, or the electrostatic interaction of ions with one another or with neutral molecules or charged molecules
– force between 2 dipoles
– not a physical bond, occurs between 2 dipoles
– shift things around to get the + and the – charges together so they can stick together
Dipole
separated charges
Electrostatic Interactions
ionic interactions between + and – charges
Tertiary structure: Domains
– critical concept for larger proteins
– distinct region of a protein
–> domains can often fold independently
–> domains provide structure and/or function
– many proteins are made up of connected domains
– related domains are often found in different proteins (not made up in the exact same type of sequence, made up in a similar sequence)
– domains predict what the protein can do
– evolution has “mixed and matched” domains
Kinase Domain
– substrates an ATP to do the job
– ATP and substrate get in so the substrate can be phosphorylated
Is it better when the domain is open or closed? Why?
– more active when the domain is CLOSED
– when is it open, there is a loop that blocks the ATP binding site
– when it is closed (activated), the 2 domains fold in and the loops moves out and the substrates can get in to be phosphorylated
Quaternary Structure
Both Tertiary and Quaternary structures: involve hydrogen bonds, disulfide bonds, depends on multiple weak interactions, can be disrupted by exposure to denaturing agents (does NOT involve interactions between different polypeptide chains)
Protein Subunits (Quaternary structure)
– proteins that consist of more than 1 polypeptide
– forces/bonds involved are the same for tertiary (hydrophobic, hydrogen bonds, Van der Waals, ionic bonds, disulfide bonds)
– repeating pattern every 7 residues: abcdefgabcdefg…
– every “a” and “d” position is hydrophobic
– every “e” and “g” position is usually a charged amino acid
– charged because one of the polypeptides has a hydrophobic red “stripe” (on one side of the helix) that stick the 2 polypeptides together because they want to get away from the aqueous solution
Examples of proteins containing coiled coils
1. EB1: binds ends of microtubules (transfers things up and down- neurons)
2. GCN4: transcription factors
Wire protein structure
– side chains and proximity
– predict amino acids involved in function
Ribbon protein structure
– visualize secondary structures
Space-filling protein structure
– gives a feel for shape
– which amino acids are on the surface
– predicts interactions with water or proteins
Post-translational modifications
1. lipoproteins bind lipids
2. metalloproteins bind metal ions
3. hemoproteins have an attached heme group
Post-translational modifications:
1. Alters stability or signalling (4)
1. Phosphorylation
2. Ubiquitination
3. Acetylation
4. Sumoylation
Post-translational modifications:
2. Alters location (2)
1. Myristylation (lipid/fat)
2. Farnesylation
Ubiquitination
– gets targeted for 26S, where it gets degraded
– not all has to get degraded, could be monoubiquitination, which could change their function
Acetylation
– changes protein function within the cell
What kind of post translational modification would be needed to cross the phospholipid bilayer?
– hydrophobic, or fatty acid to target it up to the phospholipid bilayer
– hydrophobic interactions make a secondary structure, which can turn into a tertiary structure, etc (one process leads to many things)
Protein Folding
– folding pathways are likely specified by sequence
– co-operativity: thousands of weak interactions
Molecular Chaperones
– proteins that help with folding
– probably a stepwise process (hydrophobic interactions, then secondary structure, then tertiary)
– bind to partially folded polypeptide chains to help such chains attain their tertiary structure
– often involved in the movement of proteins across cellular membranes
– protect unfolded polypeptide chains from cleavage by proteases
– prevent newly synthesized polypeptide chains from associating with the wrong partners
– are usually high molecular weight ligands which bind to proteins
Conformational Flexibility and Changes
– proteins are not completely rigid structures
– various types of motion is possible depending on the structure
– often domains can move, connected by flexible linkers (called conformational changes)
– 2 domains are open, once glucose comes in, it binds and then the 2 domains close (flexible)
Denaturation
– when extreme conditions cause unfolding
– breaks weak bonds that stabilize structure
– involves the disruption and possible destruction of both the secondary and tertiary structures
– since denaturation reactions are not strong enough to break the peptide bonds, the primary structures remain the same
(see note for figure)
What causes denaturation to occur? (7)
1. pH
2. high temp
3. alcohol
4. heavy metal salts
5. detergents/certain small molecules
6. urea
7. guanidine hydrochloride
Protein Thermal Irreversible Denaturation
once you fry an egg, you can’t go back
Protein Families
– new proteins come from old ones
– proteins are related by evolution
– have similar primary sequence, structures, functions, and domains
Conserved Residues
– amino acid residues that are necessary for function are found in all members
– have important functions
– ex. steroid hormone receptors
Categories: Biochemistry