Unit 1: Proteins
Covers Slides from PPT 1 to 3-16.
Sidechains differ in: size, shape charge, hydrophobicity and reactivity. They are
classified by solubility in water or polarity of the side chain.
Hydrophilic is charge polarized and capable of hydrogen bonding with water
Hydrophobic are not polarized and unable to form hydrogen bonds with water, so
water repels them in favor of bonding with itself.
Made up of amino acids (20 kinds). By convention, the left end is N terminus, right
end is C terminus. Proteins tend to have hydrophobic amino acids on the interior, to
form a core. The protein conformation is the 3d arrangement of the polypeptide.
A single amino acid consists of an alpha carbon attached to four substituents, H,
carboxyl group, amino group and R group. They polymerize through peptide bonds
which are reactions between the ionized states of amino acids undergoing
There are 4 special amino acids: Cysteine, Proline, Glycine and Histidine. They’re
special properties are: ability to form disulphide bridges (covalent bonds), rigid
structure forms kink, small size, shifts from positive/neutral on pH.
The linear arrangement/sequence of amino acids, determined by the gene. A
polypeptide of length n has 20 arrangements.
Conformation of a portion of the polypeptide resulting from folding of localized parts.
Local interactions are non covalent bonds and include: Ionic bonds, hydrogen bonds,
van der waal forces and hydrophobic effect.
There are 3 major localized structures we observe: α-helix, β-sheet and turns/loops.
The α-helix consists of carbonyl oxygens bonded to the amide nitrogen four residues
towards the C-terminus, forming a rigid cylinder. In this arrangement the side chains
are facing outwards and are responsible for hydrophobic/hydrophilic qualities.
The β-sheet consists of laterally packed β strands (5-8 amino acids long) with H-
bonds between carbonyl and amino groups of backbone in adjacent β strands.
Forms independently of properties of side chains and has two arrangements; parallel
and antiparallel. The R-chains determine the hydrophobic/hydrophilic quality of the
surfaces of the sheets. β turns connect segments of β sheets and are 3-4 AA residues. Carbonyl of AA1 is H
bonded to amino of AA4, proline at AA2 to introduce bend and glycine at AA3 to
minimize steric hindrance.
Motifs are particular combinations of secondary structures that recur in a variety of
proteins and exhibit a particular 3d conformation associated with a particular
Coiled-coil motif: 2 or more amphipathic α-helices wrapped around one another.
Heptad (7) repeats with hydrophobic residues at position 1 and 4 of repeat. Found in
DNA binding proteins.
Zinc Finger motif: Consists of an α-helix and 2 β-strands held in position by the
interaction of precisely positioned Cys or His residues with a zinc atom. Found in
DNA binding proteins
Helix-loop-helix motif: 2 α-helices joined by a loop region. Loop region can bind Ca 2+
or DNA via carboxyl side chains from Asp or Glu in the loop. Found in a vast number
of Ca binding proteins.
β barrel motif 4-10 antiparallel β-strands connected by hairpins each strand is
successively added to the previous strand until the last strand is H bonded to the first
strand to complete the barrel.
Many proteins contain regions that lack a defined conformation; Intrinsically
unstructured proteins which have a lack of tertiary structure as isolated subunits.
Bioinformatic predict that a significant fraction of the genome codes for unstructured
proteins, and that the fraction increases with the complexity of the organism.
Unstructured proteins may acquire structure when associated in a complex. Or,
unstructured proteins may assume specific folding patterns when associated with
other molecules e.g. DNA associated with a Zinc-finger complex
The overall conformation of a polypeptide; the fundamental unit of a tertiary structure
is the domain. A domain is a substructure produced by any part a polypeptide chain
that can fold independently into a compact stable structure. These are subdivided
into 2 categories, structural and functional domains.
Functional domains: regions of a protein that perform a certain activity
Structural domains: regions of protein that form compact, largely independent
Assembly of a multimeric protein; a functional protein composed of multiple
polypeptides. Note prefixes such as homo/hetero/di/tri -mer.
Acetylation: Protects against intracellular protease degradation (80% of proteins)
Methylation: Regulation of protein activity; methylation of histone tails (chromosome
structure and gene regulation) Phosphorylation: covalent transfer of a phosphate group from ATP to the -OH group
of serine, tyrosine, or threonine (kinases) to activate/deactivate proteins. Reversal of
effect by removal of phosphate group (phosphatase)
Hydroxylation: alter protein structure and function; animals - collagen - formation of
triple-stranded helix; plants - cell wall proteins
Glycosylation: Addition of carbohydrates/sugars.sugars added to -OH groups of
serine and threonine. Many secreted and membrane proteins are glycosylated - this
occurs in the Golgi apparatus. Allows for proper folding, protection from proteolysis,
sensitization of responses of cell-surface proteins
Carboxylation: allows binding of inorganic ions
Lipidation: Addition of lipid molecule; anchors proteins to membranes. Prominent of
cell signaling events.
Polypeptides can begin to fold secondary and tertiary structures while being
synthesized. Every polypeptide has a single lowest energy state, the native state.
This is the most thermodynamically favorable state. Protein folding is spontaneous
reversible and unique and all the information required for protein folding is contained
in the primary sequence.
In an experiment a 8m urea (breaks H & hydrophobic bonds) + mercaptoethanol
(breaks disulphide bridges) was added to a folded protein. Dialysis removed the
denaturants and the renaturation of the protein in vitro occurred spontaneously.
Proof that information for protein folding lies in its sequence.
Responsible for a variety of diseases, in 2 categories. Hereditary disorders and prion
based diseases. If the protein is misfolded it may be in the wrong location to
function, have no function or be detrimental to the cell.
Prion: infectious agent that is protein based. A prion is thought to be a misfolded
version of a functional protein where the misfolded protein binds to the regular
protein and causes the regular protein to assume the abnormal conformation.
Protein Folding Pathways
Most proteins fold rapidly into their correct configuration. Incompletely folded
proteins are helped to fold by chaperone proteins. Misfolded proteins are degraded.
There are two kinds: molecular chaperones and chaperonins.
Molecular chaperonins selectively bind to hydrophobic amino acids that are exposed
in the non-native conformation allowing it to fold correctly. HSP70 in cytosol and
mitochondria, BiP in ER, DnaK in bacteria. HSP70 binds to hydrophobic elements to
prevent aggregating and incorrect conformations from being formed, helping the
polypeptide to get to the correct conformation. It only moves the protein into the right
direction. HSP70 is produced in stressful environments, mostly heat. Functions by binding to protein, while hydrolysis of ATP to ADP to facilitate attaching. Protein folds
a bit, then ATP rebinds to HSP70 causing it to release, protein finishes folding.
Chaperonins form large cylindrical isolation chambers into which misfolded proteins
are fed and folded within the chamber without interference from other
macromolecules. TCiP cytosol, GroEL bacteria or chloroplast and Hsp60 in
mitochondria. Assist in folding 15% of proteins (mammals).
Misfolded protein is initially captured by hydrophobic interactions with rim of GroEL.
ATP and cap (GroES) bind. Conformational change, space enlarges, releasing
polypeptide into space and enclosing protein. ATP hydrolysis to ADP causes protein
to eject. This ejection is a timed event, and the chamber does not interact with the
protein. When ATP is bound, GroEL is in it’s relaxed conformation, however 1 is
used per subunit, making it an energy intensive process.
Intracellular proteins are selected and marked for degradation by a protein degrading
cellular machinery called the proteasome. Cells degrade: misfolded proteins,
denatured proteins, too highly concentrated proteins, proteins taken up into the cell,
and regulate levels of cyclic proteins.
Tagging of the protein of ubiquitin molecules, a small protein with 76 residues.
Degradation of the tagged protein into short peptides (7-8 residues) by the
Step 1: Ubiquitylation
Addition of Ubiquitin to a protein targets that protein for degradation by proteasome.
3 Enzyme System
E1 Ubiquitin activating enzyme
E2 Ubiquitin conjugating enzyme
E3 Ubiquitin Ligase: large family of protein each member recognizes a different
Ubiquitin activated by linkage to E1, activated ubiquitin is transferred to cys on E2,
E2 complexes with E3, E3 recognizes substrate and transfers ubiquitin to lysine side
chain of target substrate. Protein is polyubiquitinated.
Step 2 Proteasome Degradation.
Proteasome = protein degradation machine. Once a polyubiquitin tag is attached, the
protein is unfolded and then fed through one end of the proteasome cap, through the
core, and it comes out the other. The core makes cuts at roughly at every 7-9 AA