Biochem exam 2 Flashcards
- Spatial arrangement
Conformation
Native – folded – lowest Free Energy [G]
- Conformation is stabilized primarily by:
tendency to ‘bury’ hydrophobic groups in the interior of the molecule
maximization of hydrogen bonds
- Structure of the peptide bond:
6 atoms: NCC NCC
constrains the protein to certain, allowed conformations
planar
- Secondary structure
α-helix
- right-handed
- stabilized by H-bonds that are aligned roughly parallel with the helical axis
- side chains point out and are perpendicular to the helical axis
- Ala & Leu are strong helix formers
- Pro & Gly are helix breakers. Pro forms a kink
β-sheet
- created by the planarity of the peptide bond and tetrahedral geometry of the α carbon
- held together by the H-bonds between amides and carbonyl groups
- side chains protrude and alternate
- T, V, I, Y, F, and W are found in β-sheet
parallel β-sheet
- H-bonded strands run in the same direction
- results in a bent H-bond which is weaker
- individual strands can be close or distant in the primary structure
anti-parallel β-sheet
- H-bonded strands run in opposite directions
- results in linear H-bonds which is stronger
type 1 β-turns
- find Pro
type 2 β-turns
- find Gly
strands and sometimes helices will have arrows to indicate N and C-terminal of the secondary structure
- Tertiary and Quaternary structure
Major groups: fibrous and globular proteins
Examples of fibrous proteins (know general structural features and properties):
α keratin
- strong
- α-helix
- R-H helix, L-H super-helix (R-H alpha helices wrapped into an LH coiled-coil, stabilized by crosslinked covalent disulfide
- S-S- cross-links
- hair, hooves, nails, horns, outer skin
collagen
- gives tensile strength
- in connective tissues, which are tendons, cartilage, organic matrix of bone, cornea
- strong
- L-H helix and R-H super-helix
- 3 strands
- novel x-links
- each protein folds into an LH helix called alpha chains, not helices
- structure: 3LH helix (which are alpha chains) twisted in a RH manner to give strength
- can find G, A, P HyPro, HyLys
silk fibroin
- anti- parallel β sheets, linear, run in opposite directions, stronger
- fully extended – not stretchable
- no covalent x-links
- rich in A, G
- fully extended - no stretching
- has lots of H-bonding + van der waals but no covalent X links so it is flexible
Myoglobin structure – features: largely α-helical, buried hydrophobic groups, heme
globular
binds to Fe, & O2
in muscles
related to hemoglobin but has more affinity for O2
8 alpha helices connected by loops
heme is a porphyrin ring with Fe in the center
prosthetic group (non-protein forming part): heme
proximal histidine is directly attached to the Fe, a distal histidine group hovers near the opposite face
hydrophobic groups are buried
- Methods of determining 3-D structure:
X-ray crystallography
NMR spectroscopy
Artificial Intelligence to predict 3-D structure.
X-ray crystallography
pros
- no size limits
- high resolution
- well established
cons
- hard for membrane proteins
- have to crystallize
- not dynamic so do not get full protein made. May not represent reality
NMR spectroscopy
pros
- no need to crystallize
- dynamic studies are possible
- interactions with ligands- best for small proteins
cons
- hard for insoluble proteins
- best for small proteins
Motifs and Domains
motifs:
- stable arrangement of several secondary structures elements like: alpha helix, beta helix
- usually within a larger protein
- not functional outside of larger protein
- AKA super-secondary structures
- can be found in numerous proteins
- proteins are made of different motifs folded together
- motifs exist in larger proteins and do not retain structure or function when separated from large protein
domains:
- are a region of a larger protein but not in a larger protein and thus can retain structure or function when separated from large protein
- can have 1 or more motifs
- a single protein can have several domains with each one having a certain function
- Other Globular proteins: know what Motifs and Domains are, and be able to recognize them in a picture of a structure.
motif
- within a larger molecule
- beta-alpha-beta
domain
- separated from protein
- Quaternary Structure:
Example = Hemoglobin - 4 polypeptides
2 or more polypeptides associated together
- these polypeptides are associated via. a side chain side chain interactions - polypeptide backbone
what drives this association
- stability: reduction of surface to volume ratio
- genetic economy + efficiency
- bringing catalytic sites together
- Know Denaturation, Renaturation and Folding (and the proteins that assist folding); disorder, flexibility
dentauration is the loss of structural integrity + function/activity
denaturation is due to: strong acids or bases, organic solvents, detergents reducing agents, [salt], heavy metal ions, temp., mechanical stress
renaturation: break/reform kneading dough
- Folding defects and disease
Not all proteins can fold by themselves
< 100 AA fold autonomously
> 100 AA need assistants from other proteins (ha!) to fold correctly
Some proteins require other molecules – chaperones to promote correct folding.
For example:
- Hsp70 (Heat Shock Protein 70) family protects unfolded proteins from denaturation and aggregation
- Chaperonins promote correct folding
- Isomerases make sure we have the correct stereochemistry
PDI – Protein disulfide isomerase
PPI – Peptide prolyl cis-trans isomerase
normally, misfolded proteins are fixed (remodeled) or degraded (many cellular pathways for this, such as unfolded protein response)
defects in any of the cellular systems (ex, genetic defects) may affect the degree of protein misfolding
alzheimer’s:
The native (correctly folded) β-amyloid is a soluble globular protein which is critical for neuronal growth, survival, and post-injury repair
In Alzheimer’s disease, it is clipped from the cell membrane, fragmented, and then, it misfolds
This misfolding promotes aggregation
Correctly folded helices are lost, and peptides form β strands, β helices, and β sheets now insoluble
These insoluble plaques collect around the neurons and disrupt their environment and connectivity
Parkinson’s Disease, Lewy-Body Dementia: misfolded α-synuclein forms aggregates of Lewy Bodies
Huntington’s Disease: genetic mutation that increases CAG repeats (increases #of amino acids) causes misfolding and aggregation neuronal death
- Know key terms & concepts: Ligand, Binding Site, flexibility, regulation, enzymes
ligand: a molecule reversibly bound
binding site: specific location of protein
flexibility: very important (bretahing_ induced fit
regulation: of binding s important
enzymes: substrate, catalytic (active) site
- O2-binding proteins: Myoglobin & Hemoglobin: Be familiar with their structures
Myoglobin (Mb) is a tertiary structure protein
Myoglobin is a compact globular protein composed of a single polypeptide chain (153 amino acids in length)
Mainly α helices; there are 8. Also some intrinsically disordered regions (flexibility)
Carries and stores oxygen (poorly soluble)for muscles
Contains a heme prosthetic group
Histidines interact with heme and O2
Sterically inhibits oxygen from bindingperpendicularly to the heme plane (specific!)
Shaped/folded to form a “cradle” thatnestles the heme prosthetic group
- Protects iron from oxidation (free radicals bad!)
In free heme, carbon monoxide (CO) binds 20,000x better than O2
Why? “smagic” (science magic – stuff about HOMOs and LUMOs)
In Mb, heme binds CO only 40X better than O2
The protein structure acts as a gate.
The effect of histidine (His E7), forces ligands to bind at an angle.
Significantly improves O2 vs CO binding
Why not evolve Mb to bind O2 more preferentially and tightly relative to CO?
Myoglobin (Mb)
1 subunit
O2 storage
1 heme group
Hemoglobin (Hb)
4 subunits – 2α and 2β
O2 transport
4 heme groups
(Tense State
No O2 bound
Salt bridges rigid
more stable in absence of O2, and lower affinity for O2
(Relaxed State
O2 bound
β subunits interact
more stable in presence of O2, higher affinity for O2
Recall that Hb has 4 O2 binding sites
Hb is an allosteric protein: having more than one conformation; binding at one site affects the affinity of another site)
This opens the door for something called cooperativity
With the binding of each O2 molecule, (which changes the conformation of the binding subunity from T to R), the affinity of the whole protein for O2 increases.
Positive Cooperativity: binding of a ligand increases the binding affinity of subsequent ligand
Negative Cooperativity: binding of a ligand decrases the binding affinity of subsequent ligand
+ Know what θ, Ka, and Kd are, and their mathematical relationship
Kd = 1/Ka
θ = ([L]/Kd + [L])
+ Know binding affinity of Hb as a function of pO2 - sigmoid binding curve
Due to cooperativity, the curve is sigmoidal
low binding affinity for O2
One binding site = hyperbolic
Insensitive to small changes in [O2]
Mb can’t be cooperative. Why? One binding site.
Hb has a sigmoidal curve. Reflects a transition from low-affinity to high-affinity binding. This makes Hb highly sensitive to changes in [O2].
+ Know about the 2 conformations of Hb (R and T) and their different affinities for O2
(Tense State
No O2 bound
Salt bridges rigid
more stable in absence of O2, and lower affinity for O2
(Relaxed State
O2 bound
β subunits interact
more stable in presence of O2, higher affinity for O2
+ Understand Cooperative binding,
Positive Cooperativity: binding of a ligand increases the binding affinity of subsequent ligand
Negative Cooperativity: binding of a ligand decrases the binding affinity of subsequent ligand
+ Understand the Bohr Effect: effect of H+ and CO2 on Hb’s affinity for O2
Hb can also bind H+, the binding site is different from O2 or CO2 binding site
the [CO2] also influcnes the [H+]: H+ is formed when CO2 recats with Hb or H20
when Hb binds H+, its affinity fro O2 decerases
Hb is the main buffer system in RBCs
the affinity of Hb for O2 is inversely proportional to amount of H+ and CO2 bound
consequences
- peripheral tissues: High [CO2] and [H+]: low affinity
- lungs: low [CO2] and [H+]: high affinity for O2
at lower pH (more H+), there is less affinity for O2
- this is why you hyperventilate when you exercise a lot because your muscles produce lactic acid whch lowers the pH in your muscle causing a decreased affinity for O2 so you need to compensate for that decreased affinity by breathing a lot!
+ Adaptation to high altitude; effect of BPG on the affinity of Hb for O2
initially, at lower pO2, in lungs, affinity of Hb for O2 is reduced; so less is delivered to peripheral tissue (30% vs. 38%)
then, within hours, concentration of BPG increases from ~5mM to ~8mM
affinity for O2 in the lungs is reduced slightly, but in peripheral tissue more significantly; so, even though less is bound initially, more of it is released in the peripheral tissue; result: ~37% of bound O2 is delivered to peripheral tissue
so BPG lowers the affinity that Hb has for O2 and this allows Hb to drop off more O2 to the tissues!
- Cofactors & coenzymes; prosthetic group
cofactors help enzymes function
Coenzymes are often vitamins and essential toour diet.
prosthetic group is the non protein forming part of the enzyme
- Holoenzyme, apoenzyme
Holoenzyme: apoenzyme (inactive) +cofactor/coenzyme/metal ion (prosthetic group)
apoenzyme: the protein part of an enzyme
- Modifying groups: phosphoryl-, glycosyl-
Enzymes can be regulated: enzymes can beactivated (phosphorylated) or inactivated (de-)
- Biomolecules are stable because bio-reactions are slow – they need to be catalyzed
A chemical reaction occurs when colliding molecules possess a minimum amount of energy called the activation energy (Ea)
In biochemistry this is called the free energy of activation ∆G‡
Activation energy is the difference between energy levels of the ground state and the transition state (typically higher energy than both ground states)
The rate of a reaction relates to activationenergy.
A higher ∆G‡ corresponds to a slowerreaction.
∆G is the energy difference between substrateand product! NOT CHANGED BY ENZYME
- Kinetics: Enzymes affect rate (not equilibrium) by lowering activation energy
Enzymes LOWER activation energy!
A chemical reaction occurs when colliding molecules possess a minimum amount of energy called the activation energy (Ea)
In biochemistry this is called the free energy of activation ∆G‡
Activation energy is the difference between energy levels of the ground state and the transition state (typically higher energy than both ground states)
The rate of a reaction relates to activationenergy.
A higher ∆G‡ corresponds to a slowerreaction.
∆G is the energy difference between substrateand product! NOT CHANGED BY ENZYME
- Active Site, Substrate
Active Site: location in the enzyme where thereaction occurs
Substrate: substance acted upon (specific!)
- Slowest step is the rate-limiting step
The activation energy “hill”, therefore, is the rate-limiting step!
- Enzymes typically increase rates by 105-1017-fold
yeah
- B.E. contributes to catalysis by:
(1) reducing entropy,
(2) desolvation,
(3) compensating for distortion,
(4) induced fit – change in enzyme conformation
