packing Average density of 0.74 for protein
Definitions for orders of protein structure, including supersecondary and domains
1. Primary: sequence of amino acids by peptide bonds to form polymer
2. Secondary: Regular recurring arrangements of primary protein, which is polymer
a) The helices formed by the polymer backbone
b) Example: Alpha helix and Beta Sheet
Supersecondary-motif, short range associations of secondary structural elements, often
through
side chain interactions
Domain- associations of lower order structure to form a 3-D unit
3. Tertiary: folding of all helical units, which is secondary, to produce the complete 3-D
polypeptide structure. (Actual protein)
4. Quaternary: interaction between protein monomers to form multiple proteins.
Planar structure and cis/trans configuration of peptide bond unit; reason for and
consequences of
- Usually found in trans configuration because the true electron density is intermediate
- Cis peptides are energetically extremely unfavorable because of steric clashes between
neighboring C atoms
- Double bond character causes the six atoms of the peptide bond group to always be planar
Definition of and angles; angle of pro is fixed
phi controls the C'-N distance, apprx 15 degrees pucker out of planarity due to partial double
bond nature, doesnt change
psi controls the C-C distance, can rotate around the bond
angle of pro is fixed at 60 degrees because it contains a five membered ring helix
breaker( because of fixed structure
What a Ramachandran plot shows; why glycine can be located anywhere on the plot
shows the sterically reasonable values of the angles & . pg 138 for picture
Glycine is excluded because it has a wide range of angles permitted because of lack of side
chain, often found in flexible regions with unusual backbone conformations
If number on plot is negative, left handed, positive=right handed
Plot is surface of a sphere thats been sliced so 2D
Why helix and conformation predominate (i.e., because they represent minimum free
energy states)
- Proteins adopt most stable tertiary structures possible (those of lowest free energy)
- Fold as so to bury hydrophobic side chains and leave charged groups on surface
a. Since sidechains of hydrophobic residues are located in the hydrophobic core, the
mainchain atoms of the same residues in most cases are also within the hydrophobic core
b. Since the presence of polar groups in hydrophobic environment is very unfavourable,
the main chain N- and O- atoms have to be neutralized by formation of hydrogen bonds
c. The two most efficient ways of hydrogen bond formation is to build an alpha helix or a
beta sheet
Why parallel sheets are in the interior of proteins and are less stable than antiparallel sheets (viz.,
less favorable skewed H-bonds cant compete with H-bonding to water)
-Antiparallel sheets are usually arranged with all their hydrophobic residues on one side of the sheet. This
requires an alternation of hydrophilic and hydrophobic residues in the primary structure of peptides
involved
To recognize the pattern of hydrophobic residues alternating with hydrophilic residues in
polypeptide strands in antiparallel sheets at the surfacew of proteins
-All of the hydrophobic residues are arranged on one side of the sheet, thus requiring alternating
in the primary structure of the peptides
-In the polypeptide sequence there are large stretches where every other residue is a glycine
-All glycine end up on one side of the sheet and the other residues on opposite side
To recognize the pattern of Gly-X-Pro or Gly-Pro-X found in collagen strands
- Polyproline II helix strands are very extended compared to alpha helix, .936 nm/turn. 3 folded
left-handed helix (3 residues/turn)
-polyproline or poly(Gly X Pro) or poly (Gly Pro X), proline every third
-Tropocollagen- basic unit; 3-lefthanded intertwined polypeptide (polyproline II helix) chains
(1000 residues)
-forms right handed coiled-coil superhelix, forms fibrils, arrange in staggered, collagen
-unique AA composition, including hydoxylysine and hydroxylproline (HyP)
-Hydroxylproline formed by Vitamin C- dependent prolyl hydroxylase reaction
- In the triple helix, every third residue faces or contacts the crowded center of the structure, only
Gly can fit. Staggered structure so that Gly residues from the 3 strands stack along the center of
the triple helix and the Gly from one strand lies adjacent to an x residue from the second stand
and to a y from the third.
- Formation of interchain H bonds with Pro and HyPfurther stabilizes structure
Five types of supersecondary structure, including helix coiled-coil (e.g. leucine zipper
with heptad repeats having leu or similar amino acid at positions 1 and 4 of 7-residue
repeats)
-helix turn helix or helix loop helix
-Hairpin beta or beta meander
-2 adjacent anti-parallel beta strands, joined by a loop
- the hairpin motif can occur both as an isolated unit or as a part of a bigger beta sheet
-Greek key beta
-most common way to connect 4 adjacent antiparallel beta strands
-interrupted with AA sequence, it bends back
-beta-alpha-beta
-convenient way to connect 2 parallel beta strands
-right handed returns, with helice about plane of beta sheet
-alpha helix coiled
-a bundle of alpha-helices wound into a super helix. Two, three, or four helical segments may be
found in the bundle, and they may be arranged parallel or antiparallel to one another
-twists the right handed alpha helix in a left handed twist in coil reduces residues per turn to 3.5
-2 x 3.5 = 7, the positions of the side chains repeat after two turns(7 residues)
-heptad repeat- in peptide sequence of a coil-coiled structure
Structures of barrels, sandwiches, and propellers as bases of domains; what
supersecondary structures they contain
Beta barrel-made of beta alpha beta repeats
-used to form bigger domains
-active sites on the top or bottom (in the loops)
Beta sandwich- made of beta sheets; two sheets with one on top of the other
-used to change directions/turns
Beta propeller- made of beta sheets; the last strand comes back over and forms the first strand in
the next blade
-used to change directions/turns
Properties of IUPs (intrinsically unstructured proteins)
-exist and function normally in a partially unfolded state, do not possess uniform structural properties but
are still essential for cellular function, nearly complete lack of structure and high intramolecular
flexibility, adopt well-defined structures in complexes with their target proteins, abundance of
polar residues and a lack of hydrophobic residues
The three types of reversible enzyme inhibitors; their distinguishing effects on Km and Vmax; and
their diagnostic Lineweaver-Burk plots
Comp- binds to same site as substrate
Noncomp- does not bind to substrate binding site and can bind to both ES complex or the
enzyme
Uncomp- binds only to enzyme substrate ES complex and slows down the rxn prob by inducing
a conformational change in the enzyme
competitive: I increases Km by a factor of {1 + ([I]/KI)}; no effect on Vmax
uncompetitive: I decreases BOTH Km and Vmax by the same factor, {1 + ([I]/KI')}
mixed (noncompetitive): I decreases Vmax by a factor of {1 + ([I]/KI')}; "pure"
noncompetitive inhibitors have no effect on Km.
How sulfa drugs and viagra work and why ethanol is an antidote for methanol or antifreeze poisoning
-Viagra serves as a competitive inhibitor. Increases cGMP levels resulting in relaxation of
smooth muscle of blood vessels. In Inhibiting cGMP hydrolysis by inhibition of
phosphodieaterase in penile tissue boosts cGMP levels, increasing penile vascular muscle
relaxation and increasing erection
-Sulfa drugs serves as a competitive inhibitor for DHPS
-Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits
the oxidation of methanol.
Other ways to diagnose type of bisubstrate reaction (i.e., ligand binding studies and
exchange reactions)
Kinetic analysis-eg Lineweaver burk plot- distinguish from single or double displacement
Substrate binding assay to distinguish between random or ordered. Binding sites and Ks
determined by scatchard plot
Exchange reactions- attempt reverse reaction in absence of one substrate to distinguish if
mechanism is single or double displacement
What scatchard plot shows
A plot of Sbound/Sfree vs Sbound
Used to determine the number of binding sites and Ks for a substrate or any other ligand.
How irreversible inhibitors work (they covalently bond to enzymes) and several examples:
organophosphorus nerve gases and insecticides, (suicide penicillin substrates)
Nerve gases and organophosphorus insecticides work by irreversibly inhibiting the breakdown
of the neurotransmitter acetylcholine by acetylcholinesterase
Penicillin inhibit enzyme glycoprotein peptidase
Enzymes catalyze reactions by lowering activation energy, not by changing G for the
reaction
The seven reasons listed in lecture notes for catalysis by enzymes
1. Stabilization of transition state intermediates
2. Proximity and orientation substrates
3. Entropy loss in ES formation
4. Destabilization of ES due to geometric strain, electronic stain, and desolvation
ofsubstrate
5. Formation of unstable covalent intermediate
a. With side chains of reactive standard amino acids
b. With prosthetic groups ie. Coenzymes: pyridoxol phosphate, thiamine
pyrophosphate
6. General acid and/or general base catalysis via proton donation or acceptance
a. By standard amino acid side chains, typically the acid and conjugate-base forms
of Glu, asp, or his, or possibly others
7. Metal ion catalysis
a. As above via general base catalysis
b. Via electronic strain due to ability to polarize bonds
The properties of NACs (Near-Attack Conformations)
Precursors to reaction transition state, Reacting atoms are in van der Waals contact and at an
angle resembling the bond to be formed in the transition state, NACs are characterized as
reaching atoms within 3.2 A and an approaching angle of +-15 of the bonding angle in the
transition state
The amino acids that can form unstable covalent intermediates during catalysis (ser, thr,
tyr, cys, lys, his, asp, glu) and the types of unstable covalent bonds formed, including Schiff
base formation with lys
unprotonated His imidazole
unprotonated -amino group
unprotonated -amino group of Lys
unprotonated thiol (thiolate anion, -S) of Cys
aliphatic -OH of Ser
unprotonated R group (carboxylates) of Glu, Asp
Schiff base formation with lys, Changes the substrate into a strong base by abstracting a proton
Characteristics of general acid and general base catalysis and the amino acids that can act
as general acid and general base catalysts (asp, glu, his, cys, tyr, lys)
-in which H or OH is created in the transition state by another molecule or group, which is
termed the general acid or general base, catalysis where a proton is transferred in the transition
state
-Hydroxide that catalyzes the reaction is generated from water in the transition state
-can increase reaction rate 10 to 100 fold
-Asp, Glu, Cys, Tyr- donate H+
-Lys accepts H+
Ways pKa values can be shifted to permit catalysis (i.e. exam 1 info)
-pKa value increase when two amino acids of similar sidechains are near each other (ex: asp and
glu)
-Because when you abstract a proton from one, pKa will increase
-residue buried in hydrophobic pocket
-another negative charge nearby
Properties of LBHBs (Low-Barrier H-Bonds) and role in catalysis
As distance between heteroatoms become smaller (<.25nm), H becomes stronger
-DECREASE in distance between H-O INCREASES bond strength
Stabilization energy can approach 60 kJ/mol in solution
-Typical H-H strength is 10-30 kJ/mol and O-O separation is .28 nm
covalent nature
Energy barrier for O decreases to exchange
Formed between two electronegative atoms with similar pKas
Important because energy released in forming LBHB can assist in catalysis
-A weak H-bond in an enzyme ground state may become an LBHB in a transient
intermediate or even in the transition state for the reaction.
-The energy released in forming the LBHB is used to help the reaction that forms
it, lowering the reaction barrier for the reaction
-Redistribute electron density in the reactive intermediate, achieving rate acceleration by
facilitation of hydrogen tunneling
-A hydrogen transfer reaction that occurs through, rather than over, a
thermodynamic barrier
Characteristics of metal ion catalysis with Zn2+ as an example
-electrophilic catalysts
-form nucleophilic attack
-do general acid/base catalysis
-zinc is often chelated with 3 side chains (eg His) and in the 4th position it can be chelated with
h2o to generate OH- at pH 7 to serve as a nucleophile
The catalytic amino acids and their roles in the serine (also applies to cysteine) and asp
proteases
Serine Proteases
-The mechanism: A mixture of covalent and general acid-base catalysis
-Trypsin, chymotrypsin, elastase: digestive enzymes, synthesized in the pancreas and
secreted into the digestive tract as proenzymes, or zymogens; all cleaves peptide chains at a
different position
-trypsin- cleaves peptides on the carbonyl side of basic amino acid (arginine or
lysine)
-Pocket has a negative charge at its bottom, facilitating the binding of
positively charged arginine and lysine residues
-elastase- cleaves peptides on the carbonyl side of small, neutral residues
-Shallow pocket with bulky residues at the opening; only small, nonbulky
residues can be accommodated in its pocket
-Catalytic Triad- His57, Asp102, Ser195- forms at the active site of the serine
proteases; polar residues
-Relies on LBHB. Donation of proton from Ser to His lowers the pKa of
the protonated imidazole ring so it becomes a close match to that of Asp
-Results in correct orientation of His
-Energy released in the formation of LBHB is used to facilitate the
formation of the subsequent tetrahedral intermediate
-Thrombin- enzyme in the blood-clotting cascade
-Subtilisin- bacterial protease
-Plasmin- breaks down the fibrin polymers of blood clots
-Tissue Plasminogen activator (TPA)- cleaves the proenzyme plasminogen, yielding
plasmin; can minimize harmful consequences of heart attack by be able to stimulate
breakdown of blood clots
-Burst Kinetics- The release of acetate is the rate-limiting step(slow step)and accounts for
the observation of burst kinetics
-Aspartic Protease Mechanism
-Enzyme possess 2 aspartic acid resides due at the active site
-Work together as general acid-base catalysis
-Functions- digestion, lysosomal protein degradation, and regulation of
blood pressure
-Cleaves peptide bonds between two hydrophobic AA residues
-Molecular dynamics simulations indicate that aspartic proteases employ lowbarrier hydrogen bonds (LBHBs) in their mechanism
-LBHB disperses electron density in the ten-atom cyclic structure,
accomplishing rate acceleration by means of hydrogen tunneling
Types and examples of protein denaturants and how they work
-heat- break intermolecular molecules
-acid/alkali- break peptide bonds; deanimate side chains of Asparagine (Asn), Glutamine
(glu)
-Urea- breaks noncovalent bonds
-Guanideine- HCL
-Surfactants-(eg SDS)-coats protein with single charge
-Polyethylene oxide-(eg tween, titron, nonidet)
-some are more reversible than others
Tm of a protein is the melting temperature, where 50% of the protein molecules are
denatured
Chaperones: catalysts for protein folding Hsp70 and chaperonin (aka. Hsp60) and how
they work using ATP hydrolysis to drive conformational changes
HSP- heat shock proteins
-Originally observed as abundant proteins in cells given brief exposure to high
temperature
-Use ATP to change conformation
Hsp70 family
bind unfolded or partially folded polypeptides with exposed hydrophobic regions,
preventing inappropriate aggregation
some also block folding of certain proteins that need to be translocated across cellular
membranes into other compartments in an unfolded state.
bind and release polypeptides in a cycle involving several other proteins (including some
from a class called Hsp40), requiring ATP hydrolysis (catalyzed by one of the component
proteins)
chaperonins
large protein complexes required for folding of some cellular proteins that don't fold
spontaneously
The GroEL/GroES complex catalyzes ATP hydrolysis as part of the cycle in which (not
necessarily in exactly this order):
a partly folded or unfolded protein with hydrophobic region binds inside the double
heptameric GroEL cylinder with a GroES at the other end,
a major conformational change occurs,
ATP binds and is hydrolyzed and ADP and the first GroES cap are released,
another GroES cap and more ATP are bound at the other end of the GroEL complex,
the bound protein progresses in its folding pathway inside the complex and more ATP is
hydrolyzed, and
the "substrate" protein is released, but could bind again if it hasn't yet properly progressed
in its folding during the first "round".
RNA enzymes are called ribozymes, which are limited in reactions catalyzed due to presence of only
OH catalytic groups on the ribose
The peptidyl transferase of ribosomes is a ribozyme