biochem exam 2 Flashcards
what consists of 3;D protein structure
secondary
tertiary
quaternary
conformation of protein
what specific thing do proteins adopt?
what are the conformation of proteins able to accomplish
what is the native fold, what can we say about its energy?
what are two interactions within a protein that can provide stability?
what is protein conformation stabilized by
what do they combat
Unlike most organic polymers, protein molecules generally adopt a specific three-dimensional conformation
Those structures are able to fulfill a specific biological function
This structure is called the native fold (properly folded, functional conformation – lowest free energy (G)
The native fold has a large number of favorable interactions within the protein for stability –”burying” hydrophobic groups andmaximizing H-bonding
Protein conformation stabilized by:
Disulfide bonds
Weak, noncovalent interactions
THESE COMBAT ENTROPY
Favorable Interactions in Protein Folding and Maintaining a Native State
Hydrophobic Effect
Release of water molecules from the structured solvation layer around the molecule as protein folds increase the net entropy
Correctly position hydrophobic side chains depending on environment
Hydrogen Bonds
- Interaction of N-H and C=O of the peptide bond leads to local regular structures such as α-helices and β-sheets
- Side chain – side chain interactions. H-bonding between R-groups, H-bonding between backbone and R-groups.
Van der Waals Interactions
Weak attraction between all atoms contributes significantly to the stability in the interior of the protein
Electrostatic Interactions
Long-range strong interactions between permanently charged groups
Salt-bridges, especially buried in the hydrophobic environment strongly stabilize the protein
Secondary (2°) Structure
Secondary structure starts with primary structure…
The peptide bond is rigid and planar, constraining the protein to certain, allowed conformations
Carbon-carbon (psi - Ψ) bonds can rotate
Carbon-nitrogen (peptide) bond (phi - Φ) cannot rotate
In theory, + / - 180° rotation for phi/psi bonds can occur,but…
Steric Hinderance will prevent some angles from occurring…
Steric Hindrance of Side Chains and Backbone Reduces Possible Rotation
The peptide bond is rigid and planar, constraining the protein to certain, allowed conformations
In theory, + / - 180° rotation for phi/psi bonds can occur,but…
Steric hindrance will prevent some angles from occurring…
Ramachandran Plot
Shows common secondary structural elements and the acceptable range of rotation
Steric hindrance prevents all but a handful of secondarystructures
alpha helix
The helical backbone is held together by hydrogen bonds between the backbone amide of an “n” and carbonyl group of the “n + 4” amino acid
Right-handed helix with 3.6 residues (R groups – 5.4 Å per turn)
Hydrogen bonds are aligned roughly parallel with the helical axis. Stabilizes.
Side chains point out and are roughly perpendicular with the helical axis
Residues 1 and 8 align and are on top of each other.
more on alpha helices
how long is the inner diameter of the helix and what is it too small to fit into
how long is the outer diameter of the helix and where can it fit into
some amphipathic alpha helices can form what
what amino acids are helix breakers, and what will it cause?
what amino acids are helix forces
The inner diameter of the helix is about 4-5ÅToo small for anything to bind inside
The outer diameter of the helix (with side chains)is 10-12 Å. Fits into the major groove of dsDNA
Some amphipathic α-helices can form coiled-coildimers (stay tuned – keratin)
α-helices cannot be formed with Pro which will cause a kink that disrupts the helix. Gly is a helix breaker too.
Not all polypeptide sequences adopt α-helical structures
Small hydrophobic residues such as Ala and Leu are stronghelix formers.
beta pleated sheets
what creates it
what holds it together
what causes the up and down directions
what amino acids are found in them
what amino acids are not found in them
The planarity of the peptide bond and tetrahedral geometry of the α-carbon create a pleated sheet-like structure (zigzag)
Sheet-like arrangement of the backbone is held together by hydrogen bonds between the backbone amides and carbonyl groups in different strands
Side chains protrude from the sheet alternating in up and down directions
Found in β-Sheets:
Large, aromatic (Y, F, W)
Branched (T, V, I)
NOT Found in β-Sheets:
G, P
Parallel β-Sheets
In parallel β-sheets, the H-bonded strands run in the same direction
Resulting in bent H-bonds (weaker)
Individual strands can be close, or distant, in the primary structure
Antiparallel β-Sheets
where do the H-bonded double strands run
what does it result in
what is the proximity of the individual strands in the primary structure
In antiparallel β-sheets, the H-bonded strands run in opposite directions
Resulting in linear H-bonds (stronger)
Individual strands can be close, or distant, in the primary structure
Diagrams and Organization of Secondary (2°) Structure
Most proteins are globular in shape as a result of frequent turns or loops in the polypeptide chain that connects beta strands and alpha-helices. 1/3 of AA residues are in turns or loops
Strands, and sometimes helices, will have arrows to indicate N- and C- termini of the 2° structural element
β turns occur frequently whenever strands in β sheets change direction
The 180° turn is accomplished over 4 amino acids
Turn stabilized by a H-bond
Type 1:
Proline
Type 2:
Glycine
Tertiary (3°) Structure
Tertiary Structure – overall 3D arrangement of all atoms in a protein
Includes long-range contacts between AA’s in a single polypeptide chain
Stabilized by numerous weak interactions between amino acid side chains
Largely hydrophobic and polar interactions
Can be stabilized by disulfide bonds
Side chain with backbone interactions are also possible
Two Major Groups
- Fibrous (elongated; structural)
- Globular (enzymes, etc.)
tertiary structure
Often have structural rather than dynamic roles and are water insoluble
Typically contain high proportions of α-helices (keratin) or β-pleated sheets (fibroin)
Underlying structures are relatively simplistic
High proportion of hydrophobic AAs
Extensive supramolecular complexes
Fibrous Proteins
We will look at 3: α-keratin, collagen, silk fibroin
α-Keratin
where is it found in the body?
what structures make it up
what shape are they
how is the coiled-coil made
how is it stablized
Hair, nails, hooves, horns, outer skin
Strong, RH α-helix; LH parallel superhelix; strengthened by cross-links
Typical right-handed α-helices
Super twisting helices, in a left-handed fashion, wrap around each other to make a very tight coiled-coil
Cross-linked by covalent disulfide bonds – stabilizes!
Collagen
what does it provide
what are examples of it in the body?
each protein of collagen folds into what direction called what and not what
how is collagen ultimately arranged and in what direction does it face?
what 3 amino acids and 2 hydroxy amino acids are essential for collagen
which amino acid and hydroxy amino acids form a cross-link and what does it provide how many variants are there in mammals
Provides tensile strength and structure (~30 types of collagen).
Connective tissue (tendons, cartilage, organic matrix of bone, cornea)
Each protein folds into a left-handed helix (α-chains; NOT α-helix)
Coiled-coil of three separate α-chains supertwisted around each other in a right-handed manner to provide strength (like a rope).
Gly, Ala, Pro, HyLys, and HyPro (essential! specific to collagen)
Lys-HyLys (Hydroxylysine) form cross-links for added strength
30+ variants in mammals
vitamin C and collagen
what is an essential component of collagen?
what enzyme converts Pro to HyPro
in the absence of what causes that enzyme to be inactivated
what does the inactivation of that enzyme cause and what famous disease is this – who is it most observed in
HyPro is an essential component of collagen
the enzyme prolyl 4-hydroxylase is required to convert pro to HyPro
in the absence of vitamin C (ascorbic acid) this enzyme is inactivated, leading to collagen instability and connective tissue problems. this is the cause of scurvy – still observed today in people who don’t consume enough fruit
Silk Fibroin
In antiparallel β-sheets, the H-bonded strands run in opposite directions
Resulting in linear H-bonds (stronger)
Individual strands can be close, or distant, in the primary structure
Rich in Ala and Gly; antiparallel β-sheets
Resulting in linear H-bonds (stronger)
Fully extended: no stretching
Extensive hydrogen bonding and van der Waals interactions between sheets, but no covalent cross-links flexibility.
Globular Proteins
what are examples of these
what can be found in them?
what is the arrangement of different secondary structural elements?
what is the general rule for protein folding
what is not the norm
what do you want to optimize
globular proteins = enzymes, transport proteins, motor proteins, regulatory proteins, immunoglobulins, etc
alpha helices and B turns, etc can all be found
arrangement of different secondary structural elements:
–compact conformation
– Folding provides structural diversity
General Rules:
Bury nonpolar amino acidR-groups
Distant segments may cometogether, but not the norm
Optimize the number of weakinteractions
Myoglobin
what are they; what elements are bound to them and where do it exist in the body
what is it distantly related to and how does it differ
what is the general rule?
from the sperm whale:
- what is the shape of the protein
- how many alpha helices
- how many amino acids
- what kind of ring is it
- what is directly attached to the iron
- what hovers near the histidine face
Myoglobin:
Iron- and oxygen-binding proteins found in the muscle of vertebrates
Distantly related to hemoglobin, but has a higher affinity (stay tuned)
Rules are followed: hydrophobic R groups are buried
from sperm whale
Myoglobin:
Globulin protein
Eight α-helices connected by loops.
153 amino acids.
Porphyrin ring with iron in the center (heme – prosthetic group). A proximal histidine group (His-93) is directly attached to the iron, and a distal histidine group (His-64) hovers near the opposite face.
Determining 3D Structure: X-Ray Crystallography
Steps
Purify the protein
Crystallize the protein
Collect diffraction data (light bending)
Calculate electron density
Fit residues into density
Pros:
No size limits
High resolution
Well-established
Cons:
Difficult for membrane proteins
The crystal form may not represent the protein in solution or in the cell
Not dynamic (cannot determine how the protein interacts with things)
NMR
Steps
Grow the protein with a NMR-active isotope (usually of Carbon-13)
Purify the protein
Collect NMR data
Assign NMR signals
Calculate the structure
Pros:
No need to crystallize the protein
Dynamic studies possible
Interaction with ligands
Cons:
Difficult for insoluble proteins
Works best with small proteins
Organization of Globular Proteins - Motifs
what are motifs and what elements are they made out of
what are they sometimes? considered as
what can motifs be found as
what are proteins made of
Motifs are stable arrangements of several secondary structure elements (and their connections):
All alpha-helix
All beta-sheet
Combination
These are sometimes considered super secondary structures
Motifs can be found as reoccurring structures in numerous proteins
Proteins are made of different motifs folded together
organization of motifs
Organization
You can find small motifs as a part of large motifs
People have determined the structure of so many proteins, so we must organize. Two classes of organization are shown here.
Domains
Domains are relatively stable, independently folded regions within the tertiary structure of a globular protein
Each domain may encompass one or more motifs and have the same combination of motifs (EF-hand)
Domains having more than 30% of their amino acid sequence in common normally adopt the same folding pattern.
A single protein can have several domains with each domain performing a different function (e.g., enzymatic, docking, regulatory, pore, membrane anchoring, etc)
Quarternary (4°) Structure
what causes the 4* structure
what do these strategies use to associate to from larger functional clusters?
What drives the Quarternary Association?
Results from the association of two or more polypeptide subunits into a larger functional cluster
These polypeptide subunits associate into a larger functional cluster via side chain – side chain and side chain – polypeptide backbone interactions
What drives the Quarternary Association?:
Stability: reduction of surface-to-volume ratio
Genetic economy and efficiency (using quarternary structure makes our genome more efficient)
Bringing catalytic sites together
Cooperativity (biological function may be regulated by complex interactions of multiple subunits)
Intrinsically Disordered Proteins or Protein Segments
what does it contain and what does it lack
what can the protein remain in, what can it have to interact with other molecules, but what may still occur
how many human proteins fit into this
how do its amino acids affect its structure?
what kind of amino acids are in this and what do they usually lack
what can disordered regions do to facilitate what with numerous what
Contain protein segments that lack a definable structure. Or, the entire protein
The protein can remain in a primary structure indefinitely, or it may have a domain that can interact with other molecules, but the rest of the protein will remain disordered.
Possibly 1/3 of all human proteins fit this designation.
They are composed of amino acids whose higher concentration forces a less-defined structure: Lys, Arg, Glu, and Pro. They usually lack a hydrophobic core
Disordered regions can conform to interact with many different proteins, facilitating possible interactions with numerous partner proteins
Protein Stability (Proteostasis) and Folding
Proteins can exist in native conformation, or other conformations; flexibility is important
Proteostasis is the constant level of the active set of proteins in a cell.
Maintaining proteostasis is a complex process, involving: synthesis, folding, unfolding, degradation, modification, etc
Protein Stability (Proteostasis) and Folding and structure
Most protein’s function depends on its 3D-structure.
To be active a protein must be correctly folded (native conformation)
The order of amino acids can help determine what this 3D conformation will be
Loss of structural integrity and function/activity is called denaturation
Proteins can be denatured by:
1. Strong acid or base
2. Organic solvents
3. Detergents
4. Reducing agents
5. Salt concentration
6. Heavy metal ions
7. Temperature
8. Mechanical stress
Think about cooking for a lot of these….
A detergent (soap) can get into the hydrophobic core and disrupt those interactions
Reducing agents will break disulfide bonds
Salt can break salt-bridges (ionic interactions)
Heavy metal ions tend to be reducing agents
Temperature can increase the instability (break) bonds
We can also physically break/reform proteins (kneading dough, tenderizing meat)
MAYBE REVERSIBLE – RENATURATION
These protein interactions are why life tends to occupy a narrow range of conditions.
Denaturation– lose structure and thus function. Ex: breaking put disulfide bonds. Order to amino acids can determine protein
Reducing agents oIL, RIG, changing elctrons changing charges, changing amount of H+ on molecule which is disput lots of this
Salt concentration: change ionic character and change side chain interactions an disrupt proteins
Heavy metal: reducing agents
Temp: melt it, ex: egg
Mechanical stress: knead dough, changing structure and breaking proteins
All of this requires knowledge of previous. Chapters
Renaturation: maybe you do not get rid of all of the hydrogen bonds
protein folding
Proteins fold to the lowest-energy (most stable) conformation in the microsecond to second-time scales.
It is not a “search”
The direction is biased towards thermodynamically and sterically possible conformations
Nonpolar inside, polar outside! (we’ve gone over this)
Folding of secondary structures can occur at multiple sites at the same time.
Then, secondary structures interact to find the lowest energy minima (conformation)
Folding often begins before protein synthesis is complete
can all proteins fold by themselves?
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
Folding Defects and Disease
what if protein is misfolded
normally, misfolded proteins are fixed (remodeled) or degraded (many cellular pathway for this, such as the unfolded protein response (UPR)
defects in any of the cellular systems (ex: genetic defects) may affect the degreeof protein misfolding
native
The native (correctly folded) β-amyloid is a soluble globular protein that is critical for neuronal growth, survival, and post-injury repair
In Alzheimer’s disease, it is clipped from the cell membrane, fragmented, and then, 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
misfolded proteins and parkinsons and huntingtons
Parkinson’s Disease, Lewy-Body Dementia: misfolded α-synuclein forms aggregates Lewy Bodies
Huntington’s Disease: genetic mutation that increases CAG repeats (increases #of amino acids) causes misfolding and aggregation neuronal death
Overview of Globular Protein Functions
- storage of ions & molecules myoglobin, ferritin
transport of ions and molecules: hemoglobin, serotonin transport
defense against pathogen: antibodies, cytokines
muscle contraction: actin and myosin
biological catalysts: chymotrypsin & lysozyme
myoglobin, ferritin and hemoglobin: store oxygen and iron
Hgb carries things
Defend: antibodies are globular proteins
Being a catalyst for a rxn ex: phosphofructokinase
*be able to know one kind from each topic
Ligand Binding
define
ligand
binding site
flexibility
regulation
substrate and catalytic (active) site
a molecules reversibly bound
specific location on protein
very important (breathing)– indices fit
regulation binding. important
Binding is via noncovalent forcesthat dictate protein structure!
Remember this!
This allows the interactions to betransient!
Illustrating Protein Function Through Oxygen Binding
O2 is critically important (obvs), but poorly soluble
Transition metals (esp. Fe) can transport O2 but can damage cells
So there is a need to sequester Fe
How? Heme!
Heme is a prosthetic group – a porphyrinring complex with an iron ion (Fe2+)
Binds oxygen reversibly
Oxygen binds via heme as amino acidscannot bind oxygen
6 coordination sites: 1-4 (nitrogen),5 (amino acid), 6 (oxygen)
Lets talk about myoglobin and binding
Find myoglobin in muscle
Hgb in red blood cells
Porphyrin ring with an iron ion that binds O2 reversibily which means that oxygen is a ligand – binds Hgb and drops it off in tissues because that is where we need it
Exercise: use O2 to make ATP so no O2 in muscle because form CO2 in the muscle and so we exhale a lot
Heme is stabilized in the Hgb in 6 places. AA that helps it is histidine
more on Ligand Binding
High specificity can be explained by the complementary nature of the binding site and the ligand
Molecular complementarity: size, shape, charge, hydrophobicity
Lock and key model: proteins and ligands have a rigid interaction with each other
Proteins are FLEXIBLE!
Induced Fit Model: both the ligand and theprotein can change conformations uponbinding; makes binding site more complementary to the ligand
Binding: non covalent forces: van der waals, hydrophobic interactions– allows to be transient. Want molecules to release from the site. Want transient interactions so we can regulate —why it cannot be permanent
Complementary forces: allows and generates high specificity
Complementary fit
Ligand can fit into the protein
drug design: binding pocket
Myoglobin (Mb)
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!)
Shape means function so for Myo. Where shape determines function, histidenn inhibits O2 from binding perpendically
Myo is shaped to nestle heme prosthetic group because heme is nestled it protects heme from getting oxidized. Cradle prevents iron from being oxidized to prevent free radical from forming
Free radicals
Histidine ensures specificity for O2
more on Myoglobin (Mb)
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?
Highest occupied molecular orbital/lowest unoccupied molecular orbital and sigma/pi bonds
Why do we transport oxygen? To let it go. If it bound even better, then O2 might not be able to dissociate as easily when it reaches the target tissues.
Heme in Myo. CO binds only 40X
Decreases specificity for heme is from 20,000X to 40X
Why CO doesn’t bind as well when….yay histidine
Protein-Ligand Binding: Quantitative
typical receptor-ligand interactions
sequence-specific protein- DNA
biotin-avidin
Remember your equilibrium constants: Keq = [products]/[reactants]
So, if P = protein, L = ligand, then the protein-ligand reaction is…
A lower Kd corresponds to a higher affinity (stay tuned)
Binding affinity
Kd: concentration of molecule where you get 50% binding
Low Kd: higher the affinity, how well and specific the binding is
High Kd: lower the affinity
Keq is adapted to Kd
Protein + ligand = proteinligand so protein ligand rxn is …
Kd = 1/Ka because they are opposite
Ka: forward direction, proteinligand is product, protein + ligand is reactant
Kd is protein + ligand / proteinligand
Protein Stability (Proteostasis) and Folding
Proteins fold to the lowest-energy (most stable) conformation in the microsecond to second-time scales.
It is not a “search”
The direction is biased towards thermodynamically and sterically possible conformations
Nonpolar inside, polar outside! (we’ve gone over this)
Folding of secondary structures can occur at multiple sites at the same time.
Then, secondary structures interact to find the lowest energy minima (conformation)
Folding often begins before protein synthesis is complete!
Local structures probably form first, followed by longer-range interactions.
It is a sequence of fold, unfold, fold, unfold, etc until we reach the lowest energy minima
chaperones and Hsp70
how many AA can fold and not fold properly?
what do other proteins need to support with folding
what does Hsp70 do
what di chaperonins do
what do PDI & PPI do
Not all proteins can fold by themselves
< 100 AA can 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
what is the disease and affected protein of the disease?
Alzheimer’s
familial amyloidotic polyneuropathy
cancer
creutzfeldt-jacob disease
hereditary emphysema
cystic fibrosis
Alzheimer’s: Beta-amyloid peptide
familial amyloidotic polyneuropathy: transthyretin
cancer: p53
creutzfeldt-jacob disease: prion
hereditary emphysema: alpha 3 antitrypsin
cystic fibrosis: CFTR
notes on ligand binding
Remember this when you’re thinking about medicinal chemistry.
If the binding were covalent, the connection/binding would be too strong for the ligand to release on its own/very easily (also would take the enzyme/receptor out of commission)
Pharmacy is molecule binding to receptor
Ligand can bind and unbind
Binding site: can be active site, allosteric site (somewhere else)
Flexbibility: proteins are dynamic due to van der waalls ect, they are not fixed so we can have iiinduced fit: enzyme changes change and chages shaepe of ligand – not really lock and key but really induced fit
Regulate: prevent, encourage, one thing bndiing one helps another bind a second time: coopoerativlity
Active site: where rxn happens in
Substrate: what is part of the rxn
How many bonds can the Fe2+ ion at the center of the heme group form?
6 bonds or coordination sites
Which of the following is true of the binding of CO by free heme, compared to the binding of O2 by free heme?
– it is 20,000x stronger BUT in Mb, heme binds CO only 40X better than O2 WHICH IS DUE TO THE Histidine in Mb
Remember your equilibrium constants: Keq = [products]/[reactants]
So, if P = protein, L = ligand, then the protein-ligand reaction is…
A lower Kd corresponds to a higher affinity (stay tuned)
What is the equation for protein and ligand
protein + ligand = protein-ligand
what is the equation for association (Ka) and dissociation constant? (Kd)
Ka = ([PL]/[P][L])
Kd = ([P][L]/[PL])
if we measured the amount of oxygen binding as a function of oxygen (ligand) concentration
as concentration increases sufficiently, the amount of binding would… this is what
theta
the amount of oxygen bound to Mb would increase with oxygen concentration
reach a maximum value, this is referred to as saturation
theta = ([PL]/[P]total
= [L]/[L] + Kd
or rearranged [L] = (theta/(1-theta)) x Kd
slope of the curve (how quickly we reach saturation) depends upon the affinity of the interactions
Total protein is protein + protein + ligand
Eqns will be given
Fraction saturation or fractional binding is theta
10 protein total but only 5 is PL, we have 5/10 = 0.50, we have half binding
