Term
| What is the mode of action of AFPs? What is the net effect? |
|
Definition
AFPs function to LOWER the freezing point of ice (make it so colder temp required to freeze)
They do so by causing ice to grow in a curved surface around them - this is energetically unfavorable, so for ice to grow lower temperatures are required |
|
|
Term
| 3 different animals AFPs can be found in |
|
Definition
Winter flounder
Spruce budworm
Snowflea |
|
|
Term
| What is the predominant aa in the sequence of the winter flounder AFP? |
|
Definition
| Ala (rich in Ala residues because of helical structure) |
|
|
Term
| What is unique about the structural levels of the winter flounder AFP? |
|
Definition
Only has primary and secondary structure
NO tertiary or quaternary
Secondary structure consists of an a-helix of approximately 10 turns |
|
|
Term
| What primarily stabilizes the structure of the winter flounder AFP? |
|
Definition
| H-bonding (b/c H-bonds are more energetically favorable at lower temperatures) |
|
|
Term
| Why is H-bonding commonly used to stabilize AFPs? |
|
Definition
| Because H-bonds are energetically more favorable at lower temperatures |
|
|
Term
| 5 stabilizing features of the winter flounder AFP |
|
Definition
Ala-rich in its primary sequence (favorable for helix)
H-bonds stronger at lower temperatures
Arg @ C-terminus, Asp @ N-terminus - compensating charges stabilize
Helix capping
Side-chain direction (Lys & Glu 4 aa's apart) |
|
|
Term
| What are two instances of dipole compensation seen in the winter flounder AFP? |
|
Definition
Arg @ C-terminus, Asp @ N-terminus (charge compensation)
C-terminus is amidated to get rid of negative charge |
|
|
Term
| What is the modification on the C-terminus of the winter flounder AFP? |
|
Definition
| It is amidated - gets rid of negative charge |
|
|
Term
| How else (besides dipole compensation) do Asp/Arg stabilize the winter flounder AFP? |
|
Definition
By HELIX CAPPING
Asp forms H-bonds with backbone NH of 3rd and 4th residues
Arg forms H-bonds with backbone carbonyls at C-terminus |
|
|
Term
| How many aa's are seen in the repeat in the winter flounder AFP? |
|
Definition
|
|
Term
| What does the helical wheel of the winter flounder AFP show? |
|
Definition
| A variable HYDROPHILIC face and a conserved HYDROPHOBIC FACE |
|
|
Term
| Difference between Ala17 and Ala19 in the functioning of the winter flounder AFP? |
|
Definition
Mutagenesis in A17 = NO functionality in protein
Mutagenesis in A19 = protein is still functional
Therefore A17 is important for function |
|
|
Term
| What does the conserved hydrophobic face of the winter flounder AFP likely confer about its function? |
|
Definition
| Since it is a conserved trait, it likely means that it is important for its function |
|
|
Term
| What is the current theory of winter flounder AFP function? |
|
Definition
The conserved hydrophobic face orders water molecules in a particular manner (hydrophobic effect)
This ordered array is similar to what is seen in ice, so these H2O moc associated with AFP quickly merge into the crystal lattice of ice and indirectly bind the AFP
Note that it is HYDROPHOBICS that facilitate binding, NOT H-bonding |
|
|
Term
| What is the # of aa's in the spruce budworm AFP repeat? What smaller pattern is seen within this repeat sequence? |
|
Definition
15-17 aa's are present in the repeating sequence pattern
TXT pattern is observed (Thr-X-Thr) |
|
|
Term
| What secondary structure makes up each of the 15-17 amino acid repeat in the budworm AFP? |
|
Definition
|
|
Term
| How many B-strands are present in the entire length of the budworm AFP? |
|
Definition
| 21 (3 B-strands x 7 repeats) |
|
|
Term
| What secondary structure is seen in the spruce budworm AFP? |
|
Definition
A B-helix!
It is a L-handed helix made up of 7 turns (1 turn/repeat of 15-17 aa's) |
|
|
Term
| How many B-sheets make up the B-helix of the budworm AFP? What types of sheets are these? |
|
Definition
There are 3 B-sheets which make up the B-helix of the AFP (sheets made of PARALLEL B-strands)
There is one B-sheet per face of the B-helix (looks like toblerone/triangular prism) |
|
|
Term
| Difference between the core structure of the winter flounder and budworm AFPs? |
|
Definition
Winter flounder - has NO hydrophobic core
Budworm - has a hydrophobic core filled w/ hydrophobic R groups |
|
|
Term
| What stabilizes the B-sheets intrinsically? |
|
Definition
The H-bonding network within them
Remember H-bonds are more favorable at lower temperatures |
|
|
Term
| What kind of interactions stabilize the B-helix of the budworm AFP? |
|
Definition
H-bonding -> intrinsically stabilizes 3 B-sheets
Hydrophobics -> hydrophobic interactions of sidechains w/in hydrophobic core
Electrostatics -> Lys/Arg paired with Asp/Glu on the EXTERIOR hydrophilic face |
|
|
Term
| What is unique about the TXT pattern of the budworm AFP and the structure of the B-helix in relation to its binding ability to ice? |
|
Definition
The face containing the TXT repeat is the FLAT FACE - face has two parallel rows of Thr residues
The Me group on the Thr acts hydrophobically to order the H2O molecules, while the -OH group of Thr forms H-bonds to stabilize
Forms a row of H2O moc in between the Thr rows on the flat face, get indirect binding of AFP to ice via well-oriented H2O |
|
|
Term
| Method of ice binding seen in the budworm AFP |
|
Definition
Flat face of helix has TXT repeat -> two rows of Thr form flat surface of face
Thr hydrophobically lines up H2O moc, then H-bonds with them to stabilize
Spacing of H2O moc. on flat face mirrors what is seen in ice -> facilitates easy binding
AFP indirectly binds to ice via the hydrophobically ordered H2O molecules |
|
|
Term
| How many faces of the B-helix is the TXT repeat seen on? |
|
Definition
| Seen on only 1 face = FLAT FACE |
|
|
Term
| What is the predominant amino acid seen in the snowflea AFP? |
|
Definition
| Gly (45% of total composition) |
|
|
Term
| What is the repeat in the primary sequence of the snowflea AFP? |
|
Definition
Gly-X-Y
X and Y are variable |
|
|
Term
| Compare the collagen and snowflea AFP's helical structures: |
|
Definition
Helical axis of AFP is straight; see a slight R-handed curvature in axis of collagen helix
NO hydrophobic core in either
H-bonding perpendicular to helical axis to stabilize in both scenarios
Pro only in turns in AFP; Pro is found throughout in collagen (contributes to long pitch seen)
Gly-X-Y structure of the repeat is common, but what occupies X and Y for collagen and the AFP differs significantly |
|
|
Term
| What is the tertiary structure of the snowflea AFP? |
|
Definition
Present as 6 L-handed helices in total; 2 rows of 3
Middle two helices (on top and bottom) have higher percentage of Gly residues so they can be packed more tightly |
|
|
Term
| Does the stereochemistry of the aa's used to synthesize the snowflea AFP affect its function? |
|
Definition
| NO, the AFP functions the same no matter whether it is made of D or L amino acids because ICE is ACHIRAL |
|
|
Term
| How does the snowflea AFP bind to ice? |
|
Definition
One of the two faces of the AFP's tertiary structure is FLAT & HYDROPHOBIC
Hydrophobic effect orders water molecules and flat face allows them to arrange to mirror array seen in ice to facilitate easy binding (similar to flounder AFP) |
|
|
Term
| What is the only AFP without tertiary structure? |
|
Definition
| The winter flounder AFP (only has secondary of a single a-helix of 10 turns) |
|
|
Term
| What are similarities seen across all 3 types of the AFPs discussed? |
|
Definition
All bind to the same ligand = ICE
All have a relatively flat & hydrophobic ice binding face (all use hydrophobic effect to bind ice) |
|
|
Term
| Where is the active site of an enzyme usually located? |
|
Definition
| In a pocket/groove of its structure |
|
|
Term
| Stereospecificity of Enzymes |
|
Definition
Need enzyme and substrate to either both be made of D or both be made of L amino acids for functionality
Enzymes are STEREOSPECIFIC |
|
|
Term
| 3 ways that enzyme activity can be regulated by ligands/changes in structure: |
|
Definition
Allosteric effectors - binds somewhere other than active site to change properties
Inhibitors - compete with S binding for the active site
Covalent modification of enzyme (phosphorylation) |
|
|
Term
|
Definition
| The FULL functioning enzyme w/ all necessary groups/cofactors attached |
|
|
Term
|
Definition
| Only the protein portion of the enzyme |
|
|
Term
| What is the relationship between the function of the enzyme and the equilibrium of the catalyzed reaction? |
|
Definition
Enzymes INCREASE THE RATE at which equilibrium is reached by lowering the activation energy for the reaction
The enzyme DOES NOT affect the equilibrium position in any case whatsoever |
|
|
Term
| Why are two different activation energies seen on a given reaction coordinate? |
|
Definition
Because it depends on the starting state (whether you start from S or you start from P)
Activation energy = energy difference between the ground state (where you start) and the highest energy point on graph (transition state) |
|
|
Term
| What is the enzyme's effect on deltaG of the reaction? |
|
Definition
It has NO EFFECT on deltaG; that simply represents the energy difference between S and P
The enzyme only alters the activation energy for the reaction (lowers energy of transition state) |
|
|
Term
| In theory, what is true about ALL enzymatic reactions? |
|
Definition
| In theory, they are ALL reversible |
|
|
Term
| How can you determine the stability of P vs. S on a reaction coordinate? |
|
Definition
| Check the relative free energy level of each state (lower free energy = more stable) |
|
|
Term
| What is energy required for in the reaction (what does activation energy do)? |
|
Definition
Need to align reacting groups on molecules
Formation of unstable intermediates
Rearrangement of bonds
Formation of transient charges |
|
|
Term
| How is the activation energy related to the rate of the reaction? |
|
Definition
High activation energy = slow rate
Low activation energy = fast rate
They are inversely proportional |
|
|
Term
| Name 2 ways in which the reaction rate can be increased: |
|
Definition
Add energy by increasing temperature or pressure of the system
Lower activation energy by adding a catalyst to provide a different reaction pathway |
|
|
Term
| Difference between transition state & intermediate? |
|
Definition
Transition state - negligible lifetime; fleeting moment of reaction
Intermediate - actual chemical entity along reaction pathway |
|
|
Term
|
Definition
| Step with the largest activation energy (biggest free energy difference between S and transition state) |
|
|
Term
|
Definition
deltaGb
deltaG of the uncatalyzed reaction minue deltaG of the catalyzed reaction
It is the energy difference between the highest energy transition state of the two different reaction pathways |
|
|
Term
| Name 4 ways in which enzymes lower the activation energy? |
|
Definition
Rearrangement of covalent bonds
Non-covalent interactions between E and S
Preferential binding of transition state
Proximity & orientation effects |
|
|
Term
| What does the rearrangement of covalent bonds allow for in an enzyme catalyzed reaction? |
|
Definition
| Gives an alternate reaction pathway -> possibility of lowering energy of transition state by this method |
|
|
Term
| What gives rise to the binding energy of a catalyzed reaction? |
|
Definition
NON-COVALENT interactions between E and S throughout their surface of contact
Interactions release energy to lower the free energy state of the system (make it more stable) |
|
|
Term
| Where is most of the energy which actually increases the reaction rate derived from? |
|
Definition
| The binding energy (non-covalent interactions in ES complex which lower energy state of system) |
|
|
Term
| What is the enzyme more complementary to - the substrate or the transition state? Why? |
|
Definition
More complementary to TRANSITION STATE
Need to be more complementary to transition state so that there is a driving force to reach it
If E were more complementary to S, then the transition state would not be reached because the ES complex would be too stable |
|
|
Term
| Different modes of enzymatic catalysis |
|
Definition
Acid-Base Catalysis - functional groups act as transient acids/bases to lower activation energy
Covalent Catalysis - rearrangement and formation of transient covalent bonds to alter reaction pathway
Metal Ion - ions used to complement negative charge, or in redox reactions |
|
|
Term
| Where does chymotrypsin cleave? |
|
Definition
After the aromatic side-chains - F, Y, W
********NOT BEFORE Pro*********** |
|
|
Term
| What is the main goal of the specificity pocket of chymotrypsin? |
|
Definition
| Position the substrate near the catalytic triad (Ser-His-Asp) |
|
|
Term
| What does Asp do to His in the catalytic triad to alter its function? |
|
Definition
| Asp deprotonates His (almost have equal sharing of H between the two) to increase its pKr value (increased basicity of His) |
|
|
Term
| What does His do to Ser to alter its function in the catalytic triad? |
|
Definition
| His deprotonates Ser (can do so because of the pKR increase thanks to Asp), to make Ser a better Nu |
|
|
Term
| What is the net effect of the H-bonding network seen in the catalytic triad? |
|
Definition
| Make Ser a better Nu so it can attack the electrophilic peptide bond to cleave it |
|
|
Term
| What are the functions of each of the 3 residues making up the catalytic triad in peptide bond cleavage? |
|
Definition
Asp102 - deprotonate His to increase its basicity; indirectly activates Ser195 via basicity of His
His57 - increased pKr allows it to activate Ser195 by deprotonating it
Ser195 - activated by His directly, and Asp indirectly, to become a better Nu to attack the electrophilic peptide bond |
|
|
Term
| What is the job of Ser195 and His57 during the catalysis of a peptide bond cleavage? |
|
Definition
Ser195 - form transient covalent bonds with substrate to alter reaction pathway
His57 - act as an acid/base in different steps of the reaction |
|
|
Term
| What happens in the hybridization of the carbonyl O during the transition state in the chymotrypsin mechanism? |
|
Definition
Goes from sp2 hybridized to sp3 hybridized - no longer planar so must be moving out of the plane
Movement of the carbonyl O out of the plane allows it to interact with the OXYANION hole in the structure |
|
|
Term
| What makes up the oxyanion hole and what is its function in the serine proteases? |
|
Definition
Oxyanion hole formed by Gly193 and Ser195
Stabilizes the transition states and tetrahedral intermediates of the catalyzed reaction by allowing the carbonyl O to H-bond with the backbone NH to stabilize it |
|
|
Term
| What does the oxyanion hole stabilize and what doesn't it? |
|
Definition
Does stabilize transition state and intermediates of reaction
Does NOT stabilize the substrate |
|
|
Term
| Differences in the specificity pockets of the serine proteases: |
|
Definition
Chymotrypsin - deep and hydrophobic
Trypsin - deep, hydrophobic w/ negative Asp residue at bottom (interact with Lys and Arg)
Elastase - has Val and Thr near the top to limit the size of the pocket (smaller hydrophobic pocket) so only small R groups can fit (G, A, S, V) |
|
|
Term
| What method of enzymatic catalysis does HIV protease use to accomplish its function? |
|
Definition
Uses ACID/BASE catalysis - Asp residues around active site act as acids/bases during reaction to increase rate
NOTE: Different than serine proteases which use COVALENT catalysis to accomplish function |
|
|
Term
| Quaternary structure of HIV protease: |
|
Definition
| A homodimer (two identical subunits) |
|
|
Term
| What is different between acid catalyzed hydrolysis of a peptide bond and cleavage with HIV protease? |
|
Definition
The SPECIFICITY of the reaction
NO specificity is observed in acid catalyzed
HIV protease specifically cleaves in between Phe and Pro residues |
|
|
Term
| What is the aim in drug design in inhibiting HIV protease from functioning? |
|
Definition
Aim for the drug to mimic the TRANSITION STATE
This is because the enzyme is most complementary to transition state -> therefore transition state shows highest affinity to enzyme |
|
|
Term
| Two different methods of controlling enzymatic activity... |
|
Definition
Control the amount of enzyme present - regulte the affinity based on concentration
Control the ACTIVITY of the enzyme (modification) |
|
|
Term
| How can enzymatic activity itself be altered? |
|
Definition
Allosteric Effectors (e.g. Calpain w/ Ca)
Modification of Enzyme (reversible, covalent; phosphorylation, acetylation)
Proteolytic Cleavage (IRREVERSIBLE) |
|
|
Term
| Example of an aspartyl protease? |
|
Definition
|
|
Term
| At which residues can enzymes be phosphorylated? |
|
Definition
|
|
Term
| What is the main difference between covalent modification and proteolytic cleavage of an enzyme to alter its function? |
|
Definition
Covalent modification = REVERSIBLE
Proteolytic cleavage = IRREVERSIBLE |
|
|
Term
| Example of an allosteric enzyme? |
|
Definition
| ATCase = aspartate transcarbamoylase |
|
|
Term
| How many subunits make up ATCase? How many active sites are there? |
|
Definition
12 subunits - 6 catalytic, 6 regulatory
There are 6 active sites on the enzyme |
|
|
Term
| What are the two allosteric effectors of ATCase, and what are their effects? |
|
Definition
ATP = positive effector; activates ATCase activity
CTP = inhibitor; inhibits ATCase activity |
|
|
Term
| Where do ATP and CTP bind to ATCase? |
|
Definition
| Both bind at the same allosteric site (NOT active site) |
|
|
Term
| In which states do the allosteric effectors of ATCase bind? |
|
Definition
T-state - inactive state; CTP binds in this state to inhibit activity
R-state - active state; ATP binds to stimulate activity |
|
|
Term
| How do the allosteric effectors of ATCase alter the enzymatic activity curve? |
|
Definition
CTP - binds to T-state; shifts curve to the right
ATP - binds to R-state; shifts curve to the left (active) |
|
|
Term
| What are the other effectors of ATCase (non-allosteric)? |
|
Definition
The substrates (carbamoyl phosphate & aspartate) are both POSITIVE EFFECTORS
Bind to R-state to activate enzyme |
|
|
Term
| What is a key factor in the rate of the standard enzymatic equilibrium? |
|
Definition
| The substrate concentration [S] |
|
|
Term
| How can you measure the rate of the reaction if [S] is constantly changing? |
|
Definition
[S] >>>>> [E]
Therefore, at initial conditions assume that [S] is constant, so can measure initial velocity |
|
|
Term
| What is the Michaelis Constant on the graph of Vo vs. [S]? |
|
Definition
The [S] at which the enzyme is functioning at 1/2Vmax
Specific for a given ES pair
Measured in mol/L |
|
|
Term
| What is the rate limiting step of the reaction in enzymatic equilibrium? |
|
Definition
|
|
Term
| What is the overall reaction rate proportional to in the equilibrium for enzymatic catalysis? |
|
Definition
[ES]
If it is low, will have a slow rate either because [S] is too low, or there is low affinity between the two
If it is high, equilibrium will shift to produce more P (increases rate of reaction) |
|
|
Term
| What is the general class of ring the heme group is made up of? |
|
Definition
Protoporphyrin ring (Porphyrin Ring)
Ring structure made up of 4 pyrrole rings (highly conjugated & planar) |
|
|
Term
| In what state does the heme group bind the Fe atom (i.e. what state is the Fe atom in to bind to the heme group)? |
|
Definition
Binds Fe2+ in the REDUCED state
Fe2+ must remain reduced so that it can bind O2 moc. |
|
|
Term
| Two characteristics of the heme group based on its conjugated nature? |
|
Definition
| It is planar and hydrophobic |
|
|
Term
| Because of its properties (i.e. hydrophobicity) where does the heme group associate in Mb/Hb? |
|
Definition
| REVERSIBLY, but tightly, binds to the hydrophobic core of the proteins |
|
|
Term
| Structural characteristics of Mb |
|
Definition
Globular, monomeric (only has tertiary structure)
Made up of 8 helical structures (A to H) |
|
|
Term
| Function of Mb and how its O2 affinity complements it: |
|
Definition
Mb binds O2 in a simplistic manner with HIGH affinity
This corresponds to its function of O2 storage in muscle where it acts as a reservoir which releases O2 only it situations of dire need |
|
|
Term
| Interactions between His residues in core and bound Heme group: |
|
Definition
His93 - on F helix; forms coordinate bond with heme Fe atom
His64 - on E helix; forms H-bond with bound O2 moc to stabilize |
|
|
Term
| What does the rectangular hyperbola on the O2 binding curve of Hb suggest? |
|
Definition
It binds O2 with high affinity (nearly 100% saturated, even at low pO2)
Binding is NOT cooperative (not a sigmoidal curve) |
|
|
Term
| Relation between sensitivity to changes in pO2 and function of Mb and Hb |
|
Definition
Mb - insensitive to small changes in pO2 -> remains high affinity to reflect STORAGE function
Hb - quite sensitive to small changes in pO2; affinity is variable depending on pH to reflect TRANSPORT function |
|
|
Term
| Structural characteristics of Hb vs. Mb |
|
Definition
| Globular, tetramer (2a and 2B subunits) |
|
|
Term
| What is similar/different between structural characteristics of Mb and Hb? |
|
Definition
Both are GLOBULAR
The tertiary structure of the a,B, and Mb subunit(s) are all similar, despite different sequences
Mb only has tertiary structure; Hb has quaternary (tetrameric) |
|
|
Term
| How do the subunits of Hb associate? |
|
Definition
First a and B come together to form 2 separate heterodimers (rigid interface)
Then the two heterodimers come together and interact to form a more flexible tetramer
Subunits interact NON-covalently |
|
|
Term
| Which state of Hb has more electrostatic interactions? |
|
Definition
|
|
Term
| Can O2 bind to both states of Hb? |
|
Definition
YES!
O2 can bind to both the T-state and the R-state, but does so with different affinities |
|
|
Term
| What conformational change occurs during the transition from the T to the R state in Hb? |
|
Definition
| See a narrowing of the pocket between the two B-subunits |
|
|
Term
| Relation of Fe atom to the plane of the heme group in the T vs. R states |
|
Definition
In the T-state, Fe is out of the plane and heme is said to be PUCKERED
In the R-state, Fe is perfectly in line with the plane of the heme group |
|
|
Term
| Why does Fe move into the plane of the heme group in the R-state? What is the subsequent effect of this? |
|
Definition
Moves into the plane so that the coordinately bound O2 molecule can come into closer proximity to the His residue on helix E that it can form an H-bond with
Causes movement of His87 and all of helix F inwards to compensate for movement into the plane -> NARROWS B-pocket |
|
|
Term
|
Definition
| One binding event enhances the likelihood of subsequent binding events; allows efficient binding and release of O2 in the body |
|
|
Term
| Based on the O2 binding curves for each of Mb and Hb, what do the curves suggest about the mechanism of binding each protein employs? |
|
Definition
Mb - rectangular hyperbola = SIMPLISTIC (non-cooperative) binding
Hb - sigmoidal curve = COOPERATIVE binding |
|
|
Term
| How could someone determine the binding mechanism of a protein and its ligand without knowing anything else? |
|
Definition
| Look at the binding curve for the association between the two -> hyperbola = simplistic; sigmoidal = cooperative |
|
|
Term
| What is one characteristic that must be possessed in ALL allosteric proteins? |
|
Definition
Must have multiple binding sites!
Allosteric effectors cannot bind to the same site as the ligand (or else they would not be considered allosteric) |
|
|
Term
| Initially, when considering O2 binding to Hb, why is binding of O2 moc NOT sequential? |
|
Definition
| It is because initially, all 4 binding sites are nearly equivalent, so binding is indiscriminate between sites |
|
|
Term
| What does n (Hill coefficient) represent in the Hill equation? |
|
Definition
Represents the SLOPE of the line of the Hill plot
The Hill coefficient expresses the DEGREE OF COOPERATIVITY between the subunits
DOES NOT express the number of ligand binding sites! |
|
|
Term
| What does a Hill coefficient = 1 imply? |
|
Definition
| That there is NO cooperativity between the binding sites (all sites behave independently) |
|
|
Term
| What is the hill coefficient in the T and R state for Hb respectively? |
|
Definition
In both states, Hill coefficient = 1 (NO cooperativity)
The only difference is that T state is low affinity, and R state is high affinity O2 binding |
|
|
Term
| What does the section of the Hill plot where the Hill coefficient = 3 represent? |
|
Definition
It represents the TRANSITION of Hb from the T-state to the R-state
At this point, are getting enough O2 binding to cause the transformational change to occur (narrowing of pocket) that accompanies the shift from T to R state |
|
|
Term
| At which point are the subunits of Hb behaving most cooperatively? |
|
Definition
| When Hill coefficient = 3 in the TRANSITION from the T to the R state |
|
|
Term
| What are the products of hydration of CO2 (equilibrium)? |
|
Definition
|
|
Term
| What occurs to the pH in the area of the reaction of the hydration of CO2? |
|
Definition
| Get a local DECREASE in pH (increased acidity) in the vicinity of the reaction as H+ are produced |
|
|
Term
| What does the interconversion of HCO3- and H+ allow for in relation to Hb? |
|
Definition
| Allows for more O2 to be picked up at the lungs and more O2 to be dropped off at the tissues (local regulation of the O2 affinity at these sites) |
|
|
Term
| What occurs at peripheral tissues with the Bohr effect on Hb? |
|
Definition
The local production of H+ stimulates H+ uptake by the Hb molecule, causes a DECREASE in the affinity of O2 (T-state)
Optimal release of O2 at the level of the tissues
Also, because of the H+ uptake by Hb, the equilibrium shifts to produce additional HCO3- to dissolve into the blood |
|
|
Term
| What does the exhalation of CO2 due to its equilibrium in the lungs? |
|
Definition
Creates a driving force to shift the equilibrium to the right to stimulate more CO2 production
Because of this get less H+ and HCO3- production (local increase in pH) |
|
|
Term
| Where does H+ bind to Hb to regulate its affinity (exert allosteric effects)? |
|
Definition
| Binds to last residue on the B-subunits (His146) |
|
|
Term
| What is unique about His146 in the B-subunits of Hb and its pKr value? |
|
Definition
| pKr is higher than expected because the His146 wants to remain protonated to stabilize the T-state |
|
|
Term
| Where is CO2 transported on the Hb molecules? |
|
Definition
Binds as the CARBAMATE group to free amino-NH2 groups on N-terminus of each subunit
(4 CO2 moc/moc Hb) |
|
|
Term
| Where is the majority of CO2 transported in the body to get to the lungs? |
|
Definition
| Majority is transported within the blood as dissolved HCO3- |
|
|
Term
| What is the function of BPG? |
|
Definition
| To greatly reduce the affinity of Hb for O2 (binds to Hb in T-state to regulate activity) |
|
|
Term
| Where does BPG bind to Hb? |
|
Definition
| In the cavity between the two B-subunits (this is the same cavity that narrows during T->R transition) |
|
|
Term
| What stabilizes BPG binding to Hb? |
|
Definition
Several positively charged residues which line the pocket in between the B-subunits of Hb (Lys, Arg)
Works because electrostatic interactions are formed between these residues and the highly negative BPG molecule |
|
|
Term
| What would be seen if the [BPG] DID NOT increase to 8mM at higher altitudes? |
|
Definition
We would lose 8% of the O2 load dropped off at the tissues
Because of the higher altitude (lower pO2), less is picked up at the lungs and the curve remains the same at the tissues therefore, LESS is dropped off at the tissues (drop from 38% released to 30% released at tissues) |
|
|
Term
| What is seen in regards to O2 affinity when the [BPG] is upregulated to 8mM in erythrocytes? |
|
Definition
See a decrease in affinity for O2 by Hb (curve shifts to the right)!
There is slightly less O2 bound at the lung, but at the tissues the affinity is GREATLY reduced to increase the amount dropped off -> regain the 38% drop-off of O2 at the tissues |
|
|
Term
| What is the difference between beta and gamma subunits of Hb seen in adults & fetuses? |
|
Definition
| Beta subunits have a higher proportion of positively charged amino acid residues than gamma subunits |
|
|
Term
| What effect does the difference in primary structure of the beta and gamma Hb subunits on its O2 binding affinity? |
|
Definition
| Because the gamma subunit has fewer positively charged amino acid residues, it binds BPG less tightly, thereby increasing its affinity for O2 |
|
|
Term
| Two main properties of H2O? |
|
Definition
Ability to H-bond
Ability to ionize (act as a weak acid or base) |
|
|
Term
| How many H-bonds form with neighbors in the cyrstal lattice of ice? |
|
Definition
4 H-bonds formed with neighbors (maximum # possible)
This occurs because of the regular bonding pattern and the low entropy state of the ice |
|
|
Term
| True or false - H-bonds have a long lifetime? |
|
Definition
| False - H-bond exist for approximately 1 ps |
|
|
Term
| How many H-bonds are formed with neighboring water molcules in liquid H2O at room temperature? |
|
Definition
3.4 bonds formed with neighbors
It is because of the increased entropy of the liquid state - harder to order the moc in directional manner to form H-bonds |
|
|
Term
| Difference between H-bond donors vs. acceptors? |
|
Definition
H-bond donors = have H covalently bound to EN atom
H-bond acceptors = have a LP of electrons |
|
|
Term
| Why does NaCl readily dissolve in H2O? |
|
Definition
Because of the negative free energy change associated with this process
As NaCl dissociates in aqueous solution, there is an INCREASE in entropy of the system as a whole (entropy of H2O moc decreases by ordering around Na and Cl ions, but overall entropy is positive) |
|
|
Term
| What is the main interaction with non-polar solutes in water? |
|
Definition
| van der Waal's forces (transient dipole formed between non-polar molecules) |
|
|
Term
| What occurs to the entropy of water as hydrophobic molecules are introduced? |
|
Definition
| The entropy DECREASES - this is because the water becomes more ordered around the hydrophobic molecules (hydrophobic effect) |
|
|
Term
| What is the hydrophobic effect? |
|
Definition
See the aggregation of NON-POLAR solutes in water to reduce the ordering of the water molecules around the solutes (relative increase in entropy of the system)
Entropic effect |
|
|
Term
| What has a weak stabilizing effect on non-polar solute aggregation? |
|
Definition
van der Waals interactions (transient dipoles form between the solutes to slightly stabilize)
NOWHERE near as stabilizing as the hydrophobic interactions themselves though |
|
|
Term
| What is unique about the ionization of a bulk solution of water? |
|
Definition
There are actually FEW molecules ionized
See a "proton shuffling effect", because ionized H2O has a short lifetime, protons shift between neighboring H2O molecules throughout solution |
|
|
Term
| Henderson-Hasselbalch Equation |
|
Definition
|
|
Term
What is the pH if...
A/HA = 0.1
A/HA = 1
A/HA = 10 |
|
Definition
If A/HA = 0.1, then pH = pKa - 1 (90% HA, 10% A)
If A/HA = 1, then pH = pKa (50% HA, 50% A)
If A/HA = 10, then pH = pKa +1 (10% HA, 90% A) |
|
|
Term
|
Definition
|
|
Term
| What is true of ALL amino acids? |
|
Definition
ALL are a-amino acids (all have an a-amino group)
Also, ALL are L-amino acids |
|
|
Term
| What two amino acids have chiral centers on their R groups? |
|
Definition
|
|
Term
|
Definition
| pI - the point at which a molecule has NO net charge |
|
|
Term
| How do you estimate the pI of an amino acid if there are 3 pK values? |
|
Definition
| Average the two closes pK values |
|
|
Term
| Why does glycine have a lower pK value than acetic acid? |
|
Definition
| Because the protonated amino group on Gly stabilizes the negative charge on the deprotonated carboxyl group; there is no compensating charge on the acetate anion |
|
|
Term
|
Definition
| aa mass - 18 (molecular weight of water) |
|
|
Term
| How can you estimate pI of a protein? |
|
Definition
| Estimate based on relative amounts of amino acids; estimate charge at a given pH based on protonation/deprotonation of side chains |
|
|
Term
|
Definition
Used to experimentally measure pI
Create a pH gradient in tube gel with immobilized ampholytes
Run electric field through the gel -> molecule will migrate and stop at its pI (because it is net neutral here, so electrical field will have no effect) |
|
|
Term
| What does SDS page measure? |
|
Definition
| The MOLECULAR WEIGHT of a protein |
|
|
Term
| The proteins which migrate the farthest on an SDS gel will be.... |
|
Definition
| The smallest (least molecular weight) |
|
|
Term
| What are the properties of SDS page which allow it to measure molecular weight? |
|
Definition
Hydrophobically binds to proteins and causes denaturation (all proteins have relatively same shape)
Binds based on the proteins size and charge (all proteins have same charge to mass ratio)
Therefore, the only thing that differs is the moc. weight of the proteins |
|
|
Term
| What is the amount of dye that binds proportional to? |
|
Definition
| The molecular weight of the protein (heavier binds more dye) |
|
|
Term
| What does SDS do to oligomeric proteins? |
|
Definition
| Causes them to denature by interrupting the hydrophobic interactions which hold protomers together |
|
|
Term
| Under what conditions are S-S bonds formed/broken? |
|
Definition
Formed = OXIDIZING
Broken = REDUCING |
|
|
Term
| What redox conditions is SDS page typically run under? |
|
Definition
| Reducing conditions (to break S-S bonds) |
|
|
Term
| What is 2D gel electrophoresis? |
|
Definition
Combination of SDS PAGE and IEF - separates proteins based on pI and molecular weight
First need to run IEF, then run proteins on SDS gel -> need to do IEF first because when SDS binds it alters the properties of the protein |
|
|
Term
| What is true about absorbance for a given random mixture of proteins? |
|
Definition
| A280 = 1 for 1 mg/mL of the protein |
|
|
Term
|
Definition
A = Ecl
A = absorbance
E = extinction coefficient (L/mol cm)
c = concentration
l = length of pathway (usually 1 cm) |
|
|
Term
| How do dyes bind to proteins (which interactions)? |
|
Definition
| Bind HYDROPHOBICALLY to proteins (bind via hydrophobic effect) |
|
|
Term
|
Definition
| Amount of protein which catalyzes conversion of 1umol of substrate in 1 minute at 25 degrees C |
|
|
Term
|
Definition
| # of enzyme units / total protein (mg) |
|
|
Term
| What does enzyme units measure, and what does enzyme activity measure? |
|
Definition
Enzyme units = amount of enzyme present
Enzyme activity = purity of the enzyme |
|
|
Term
| Solubility as a means of purification (what happens at different [salt]) |
|
Definition
@ low [salt] - compensating charges cause aggregation of proteins (electrostatic interactions) = LOW solubility
@ increasing salt - salt ions coat the proteins to make up for compensating charges and block; proteins do not aggregate and remain in solution = HIGH solubility
@ high [salt] - excess salt prevents solvation of proteins by blocking H2O molecules, get more pronounced hydrophobic effect at high salt; proteins aggregate = LOW solubility |
|
|
Term
| What effect does pH have on solubility (pH relative to pI of protein) |
|
Definition
If pH < pI = protein will be POSITIVE; repulsion = high solubility
If pH = pI -> protein will be neutral; hydrophobic aggregation = low solubility
If pH > pI = protein is NEGATIVE; repulsion = high solubility |
|
|
Term
| What types of ions are bound in cation vs. anion exchange? |
|
Definition
Cation = binds positive ions (negative ions on column; carboxyl methyl cellulose)
Anion = binds negative ions (positive ions on column; Et3NH+) |
|
|
Term
| Two ways to control ionic interactions of proteins with the chromatography column? |
|
Definition
pH -> can change charge of proteins
[salt] -> on ion exchange columns an increase causes elution; on hydrophobic columns a decrease causes elution |
|
|
Term
| What would elute faster on a cation exchange column, a protein whos pI is greater or less than the current pH? |
|
Definition
| At protein who's pI is LESS than the pH would elute more quickly - this protein would be negatively charged and be repelled from the negative charges on the solid phase of the column |
|
|
Term
| Size-Exclusion Chromotography |
|
Definition
| See porous beads as the solid phase of the column; the LARGEST proteins ELUTE first as they take the least convoluted route through the solid phase (elute in void volume); smallest proteins elute last (elute in total volume) |
|
|
Term
| What volume do the largest proteins in size-exlcusion chromotography elute in? |
|
Definition
The VOID VOLUME (smallest volume)
This is the volume OUTSIDE of the beads |
|
|
Term
| What is the ratio of Ve/Vo dependent on? |
|
Definition
The protein's shape!
Rod shaped proteins behave larger than their MW weight measured with size-exlusion would suggest as they tumble through the solid phase |
|
|
Term
| In size-exclusion chromotography, what would elute first, a rod shaped or a spherical protein of the same MW? |
|
Definition
| ROD SHAPED -> behaves as a larger protein |
|
|
Term
| What type of molecules elute the fastest in hydrophobic chromotography? |
|
Definition
| NON-HYDROPHOBIC molecules (polar molecules) |
|
|
Term
|
Definition
Have a column with a particular ligand on the beads which specifically binds to a protein eluted into the column
Elute column by releasing free ligand into the column to compete for binding with the column ligand |
|
|
Term
| What is the Edman reagent? |
|
Definition
|
|
Term
| What conditions is Edman degradation run under? |
|
Definition
ALKALINE CONDITIONS (pH > 7)
Need these conditions to make terminal amino group a good nucleophile (in deprotonated form) |
|
|
Term
| Limitations of Edman Degradation |
|
Definition
PITC reacts twice with Lys side-chains - however only get cyclization once
Limit of < 100 aa's to sequence
Often no free N-terminus (need N-terminus for PITC) - often see acetylation of N-terminus |
|
|
Term
| Cleavage sites of proteases |
|
Definition
Trypsin = C terminal side of Lys, Arg; NOT before Pro
Chymotrypsin = C terminal side of F, Y, W; NOT before Pro
Elastase = C terminal side of G, A, S, V; NOT before Pro
V8 protease = C terminal side of D, E
Asp-N = N terminal side of D, E |
|
|
Term
| Method of non-enzymatic cleavage? |
|
Definition
CN-Br
Cleaves the C-terminal side of Met residues to form a homoserine lactone |
|
|
Term
| What happens with Edman degradation at the presence of an S-S bond? |
|
Definition
| Get NO identifiable PTHaa |
|
|
Term
| Problems with amino acid composition determination with boiling on 6M HCl |
|
Definition
Breaks ALL AMIDE bonds (all Asn/Gln is converted to Asp/Glu) - can only measure total as Asx/Glx
All Tryp is destroyed |
|
|
Term
| What are the purposes of the two MS in tandem MS? |
|
Definition
MS1 - tune so that only 1 ion goes through (separate different ones based on m/z ratio
MS2 - measures m/z ratios of new fragments of the protein (after collision with noble gas molecules) |
|
|
Term
|
Definition
| Cannot distinguish Leu and Ile (SAME mass) |
|
|
Term
|
Definition
| Show 25% or more sequence similarity; this implies common ancestry between the two |
|
|
Term
|
Definition
2 similar proteins in 1 organism; usually have different cellular functions
Formed by DIVERGENT evolution |
|
|
Term
|
Definition
| Two similar proteins in DIFFERENT organisms which have the SAME function |
|
|
Term
| Example of divergent evolution in human proteins? |
|
Definition
Globin gene - diverged over time to give rise to Mb, a, B, gamma globin subunits
All are similar proteins in the same organism, but have DIFFERENT cellular functions (paralogues) |
|
|
Term
| Difference between homology seen in covergent vs. divergent evolution? |
|
Definition
Divergent evolution implies common homology (ancestry); gives rise to SIMILAR proteins
In convergent evolution, see proteins with functions that are alike, but NO SEQUENCE SIMILARITY (NOT homologous) |
|
|
Term
|
Definition
| Spatial arrangement of atoms in a protein w/ respect to one another |
|
|
Term
| What is true about the interactions in a protein in its lowest energy (most stable) state? |
|
Definition
| The number of non-covalent interactions are MAXIMIZED to stabilize this state |
|
|
Term
| Which protein interactions contribute to FOLDING? |
|
Definition
Hydrophobics (predominant)
Electrostatics
H-bonding
NOTTTTTTTTT S-S bonds |
|
|
Term
| What is the predominant interaction which determines the secondary structure of a protein? |
|
Definition
|
|
Term
| What two effects does resonance have on the peptide bond character? |
|
Definition
Creates an electric dipole about the bond (+'ve N terminus, -'ve C terminus)
Gives the peptide bond partial DOUBLE BOND character - makes it so the bond is planar
Therefore cannot get any rotation about the peptide bond |
|
|
Term
| Where are the points of rotation in each polypeptide? |
|
Definition
| At the dihedral angles (at the alpha carbons of the chain) |
|
|
Term
| Difference between PSI and PHI bonds |
|
Definition
PSI = Ca-carbonyl C
PHI = N-Ca |
|
|
Term
| Relative to the alpha carbon of one amino acid residue in a polypeptide, which side is the phi bond on and which side is the psi bond on? |
|
Definition
PHI = on the left side (N-Ca)
PSI = on the right side (Ca-C) |
|
|
Term
| What influences the phi and psi angles of a given residue |
|
Definition
| The neighboring amino acid residues |
|
|
Term
| Which is one amino acid which can exist in the cis state as well as the trans state in polypeptides? |
|
Definition
|
|
Term
| What is special about the dihedral angles in Pro? |
|
Definition
| The PHI bond (N-Ca) is locked because of the R-group structure |
|
|
Term
| Relative proportions of cis and trans Pro in a given pp chain? |
|
Definition
95-98% trans, 2-5% cis
Note: There is NO interconversion between the two |
|
|
Term
| What happens when Pro is found in the sequence of an a-helix? |
|
Definition
| Acts as a HELIX BREAKER - helix stops there! |
|
|
Term
| All of the bitch moves of Pro to watch out for |
|
Definition
Serine proteases CANNOT cut before it (trypsin, chymotrypsin, elastase)
When present in a sequence with the secondary structure as an a-helix, it STOPS THE HELIX (helix breaker) |
|
|
Term
|
Definition
| Local, spatial arrangement of the pp backbone; determined by DEFINED and REPEATED phi and psi angles |
|
|
Term
| What is the simplest arrangement of aa's in a polypeptide? |
|
Definition
|
|
Term
| Pitch & residues/turn of NORMAL a-helix |
|
Definition
Pitch = 5.4 A
3.6 residues/turn |
|
|
Term
| Which terminus do the backbone N and backbone C=O groups point towards? |
|
Definition
Backbone NH point UP towards N-terminus
Backbone C=O point DOWN towards C-terminus |
|
|
Term
| H-bonds in relation to helical axis of normal a-helix? |
|
Definition
| PARALLEL to helical axis (between backbone NH and C=O groups which are also parallel, therefore H-bonds must be also) |
|
|
Term
| Properties of STANDARD a-helix |
|
Definition
R-handed helix
3.6 residues/turn
5.4 A pitch |
|
|
Term
| What does the i to i+4 rule imply about H-bonding in the a-helix? |
|
Definition
1st 4 residues can only act as H-bond acceptors (by backbone C=O)
Last 4 residues can only act as H-bond donors (by backbone NH) |
|
|
Term
| Electric dipole of the a-helix? |
|
Definition
N-terminus = +'vely charged
C-terimuns = -'vely charged
This is because of the dipole seen about the peptide bond |
|
|
Term
| PHI and PSI angles for a-helix |
|
Definition
|
|
Term
| What is the greatest contribution to the stability of the a-helix? |
|
Definition
| H-bonding network seen throughout |
|
|
Term
Most common aa in a-helix?
Least common? |
|
Definition
Most common = Ala
Least common = Pro and Gly |
|
|
Term
| 2 reasons Pro is uncommon in a-helices? |
|
Definition
PHI bond is fixed limiting its range of motion (cannot adopt proper angle)
No backbone H, therefore cannot be H-bond donor |
|
|
Term
| What is a difference in the location of the sequence(s) that make up B-strands vs. a-helices? |
|
Definition
In an a-helix, the sequence must be continuous
In a B-sheet, the two segments do NOT need to be close in primary structure (sequence), just need to be spatially close when the protein folds |
|
|
Term
| What differentiates secondary structural elements from loops/random coils? |
|
Definition
The repeating and defined phi and psi angles which comprise the structural elements
Loops and coils DO NOT have repeating phi and psi angles in any particular way |
|
|
Term
| What are more stable, parallel or anti-parallel B-sheets? Why is this? |
|
Definition
Anti-parallel B-sheets are MORE STABLE because of the greater # of H-bonds which form in between the two strands to intrinsically stabilize the sheets
In the parallel scenario, the backbone C=O and NH do not line up as well, giving rise to fewer H-bonds |
|
|
Term
| Differences between motifs and secondary and tertiary structure |
|
Definition
Motifs have secondary structural elements and some 3D folding W/O any associated function.
Secondary structural elements on their own have no 3D folding occurring
Tertiary structure always has associated function with its folds |
|
|
Term
| What is the fastest way to connect to parallel B-strands? |
|
Definition
|
|
Term
| What is the fastest way to connect 2 anti-parallel B-strands? |
|
Definition
The B-turn
Considered a secondary structural element because of repeating phi and psi angles |
|
|
Term
| What is the shortest # of residues which can cause a 180 degree turn in the pp backbone? |
|
Definition
| 4; what is seen in a B-turn |
|
|
Term
| What is needed to stabilize a B-turn? |
|
Definition
| At least 1 H-bond formed between residues 1 and 4 of the turn |
|
|
Term
| In a B-turn, what typically are residues 2 and 3? Why? |
|
Definition
Usually are either Pro or Gly
Pro b/c the rigidity of its phi angle and side chain limitations aid with the turn of the backbone
Gly b/c it can adopt a wide range of phi and psi angles |
|
|
Term
| What are 3 stabilizing factors of the B-a-B motif? |
|
Definition
There are INTRINSIC H-bonding networks within the a-helix and the two B-strands which stabilize them
There are side-chain interactions where the helix sits on top of the parallel B-strands; interactions are predominantly hydrophobic |
|
|
Term
| How many B-strands and a-helices comprise the structure of the B-barrel? |
|
Definition
| 8 B-strands and 7 a-helices |
|
|
Term
| What connects the B-strands of the B-barrel in its folded state? Which strands are not connected? |
|
Definition
Strands 1 and 8 are NOT connected to each other; stabilized by H-bonding between the strands
The other strands are all connected by a-helices |
|
|
Term
| What is the minimum number of B-strands which can form the B-barrel structure? |
|
Definition
|
|
Term
| Why is the B-barrel considered to be a tertiary structure and the B-a-B motif not to be? |
|
Definition
Because the B-barrel has an associated function!
The center of the barrel is often a catalytic (active) site for the protein
The folding of the barrel causes catalytic aa's to come into close proximity, even though they may not be close to each other in primary structure
Structure function relationship! |
|
|
Term
|
Definition
| The overall 3D arrangement of all atoms in a SINGLE pp chain |
|
|
Term
| What is the most important interaction involved in protein folding? |
|
Definition
|
|
Term
| Functions of S-S bonds in proteins? |
|
Definition
| To MAINTAIN the stability of the folded structure (form after folding has occurred, therefore DO NOT contribute to folding itself) |
|
|
Term
| What are the interactions which MAINTAIN and stabilized a folded protein's structure? |
|
Definition
H-bonding
Electrostatics
Hydrophobics
S-S bonds |
|
|
Term
| What structural level are domains considered? |
|
Definition
| TERTIARY - have 3D structure with associated function |
|
|
Term
|
Definition
| Regions of primary structure which INDEPENDENTLY fold into a structural and functional unit |
|
|
Term
| How are domains different than motifs? |
|
Definition
Domains = have both associated structure AND function
Motifs = only have structure (3D fold) |
|
|
Term
|
Definition
| Optimizes the functioning of a protein; instead of having 3 separate pp chains which lead towards the same goal but function independently, domains are covalently bound and work synergistically to optimize function |
|
|
Term
| Characteristics of fibrous proteins? |
|
Definition
SINGLE type of secondary structure; difficult to delineate tertiary from secondary structure
Quaternary structure is often the functional unit
Arranged in long strands or sheet like structures
INSOLUBLE in H2O b/c lots of hydrophobic residues
Important for protection & support
Oligomers build in ordered fashion to reach supramolecular structure |
|
|
Term
| Examples of 2 fibrous proteins? |
|
Definition
|
|
Term
| What structures does a-keratin comprise? |
|
Definition
| Horns, nails, hair, feathers, skin |
|
|
Term
| Comparison of a-keratin helix w/ standard a-helix? |
|
Definition
a-keratin - compressed R-handed helix with a pitch of 5.1, and 3.5 residues/turn; axis twisted to L
a-helix - straight axis, R-handed, 5.4 A pitch and 3.6 residues/turn |
|
|
Term
| Primary structure of a-keratin |
|
Definition
See a heptad repeat (labeled a-g)
Residues a and d are usually hydrophobic and interact to stabilize the quaternary structure |
|
|
Term
| Secondary/Tertiary structure of a-keratin |
|
Definition
R-handed a-keratin helix with a compressed axis causing a curvature of the helical axis to the L
Pitch = 5.1 A, 3.5 residues/turn |
|
|
Term
| Quaternary structure of a-keratin |
|
Definition
COILED COIL
L-handed twisting of two a-keratin helices to form quaternary structure
Stabilized primarily by HYDROPHOBIC interactions between a and d positions of heptad repeat between helices |
|
|
Term
| What changes occur in the primary structure of a-keratin to increase its toughness (lets say compare a-keratin of hair vs. a-keratin in horns) |
|
Definition
| If it needs to be stronger, see a higher percentage of Cys residues which form S-S bonds to stabilize the quaternary and supramolecular structures to a greater degree |
|
|
Term
| What structures does collagen make up? |
|
Definition
| CT - tendons, cartilage, bone |
|
|
Term
| Primary Structure of Collagen |
|
Definition
35% Gly, 21% Pro/4-Hyp, 11% Ala
TRIPEPTIDE repeat (Gly-X-Y)
X is often Pro
Y is often 4-Hyp |
|
|
Term
| Secondary/Tertiary Structure of Collagen |
|
Definition
L-handed helix = a-chain!
See a slight twisting of the helical axis to the right (not straight)
3.3 residues/turn; 9.4 A pitch
Pitch is extended because of Pro/4-Hyp |
|
|
Term
| Quaternary structure of collagen |
|
Definition
| 3 a-chains twist together in a R-handed fashion to form a COILED COIL |
|
|
Term
| Why can the 3 a-chains of the coiled coil of collagen be tightly packed? |
|
Definition
| Because of the relatively high proportion of Gly in its structure (small nature of side-chain allows tight packing, simlar to what is seen with snowflea AFP) |
|
|
Term
| What stabilizes the quaternary structure of collagen? |
|
Definition
H-bond interactions between the 3-chains
H-bonds are perpendicular to the helical axis |
|
|
Term
| Differences in H-bonding network in collagen vs. standard a-helix? |
|
Definition
In collagen, H-bonds are perpendicular to the axis and stabilize quaternary structure
In a-helix, H-bonds are parallel to the axis and stabilize secondary structure |
|
|
Term
| What enzyme catalyzes the hydroxylation of Pro to form 4-Hyp? What is its activity dependent on, and what is the associated pathology? |
|
Definition
Prolyl 4-hydroxylase adds the OH group post-translationally to Pro
Its activity is Vit C dependent; therefore Vit C deficiency = SCURVY
Scurvy leads to CT breakdown b/c of loss of collagen stability |
|
|
Term
| Difference in folding of the pp chain in globular vs. fibrous proteins |
|
Definition
Fibrous - chain folds linearly to maintain relatively straight shape
Globular - chain folds back onto self to form globule |
|
|
Term
|
Definition
Increased temperature
Alteration in pH
Chaotropic agents (urea and guanidine) |
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Term
| What explains the steep slope seen in the protein denaturation curve? |
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Definition
| Looks like this b/c the folding/unfolding process is COOPERATIVE |
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Term
| Why are chaotropic agents highly soluble in water? How do they denature proteins? |
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Definition
Urea and guanidine are both highly soluble because of the multiple H-bond interactions they can form
Because of high concentration, compete for H2O molecules with protein, disrupts hydrophobic effect and protein unfolds |
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Term
| Why is renaturation by dialysis possible for the protein that has been unfolded? |
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Definition
| It is possible because the code for folding of the protein is determined by its PRIMARY STRUCTURE (primary structure dictates tertiary structure) |
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Term
| What was the key conclusion of the Anfinsen experiment? |
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Definition
| SHOWED DEFINITIVELY that primary structure drives the folding of tertiary structure of a protein |
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Term
| Procedure of the Anfinsen Experiment |
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Definition
Took ribonuclease A (has 4 S-S bonds), denatured with urea and used B-ME as the reducing agent to linearize
Then allowed it to renature via dialysis, allowed to oxidize to reform S-S bonds and voila, protein correctly re-folds and function is restored |
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Term
| Hierarchial Model of Protein Folding |
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Definition
| Local secondary structure forms first, then see forming of tertiary structure (3D fold via long range interactions) |
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Term
| Collapsed State Model of Protein Folding |
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Definition
Protein collapses into a molten globule stabilized mainly by hydrophobic interactions
Hydrophobic side-chains cluster in core of protein
See some secondary elements form, but R groups are by no means fixed in place |
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Term
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Definition
| Provide a place for unfolded/misfolded proteins to fold or refold in an environment that prevents degradation or aggregation |
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Term
| What is believed to aid the folding of proteins in vivo? |
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Definition
| Protein chaperones - provide a safe environment for proteins to refold where they needn't worry about degradation or aggregation |
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Term
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Definition
Accumulation of protein fibrils in hippocampus
Formed by a fragment of APP (amyloid precursor protein)
Misfolding of APP causes cleavage to give rise to I-42 fragment
This fragment can aggregate to form B-amyloid plaques and cause Alzheimer's |
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Term
| Bovine Spongiform Encephalopathy |
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Definition
Caused by a misfolded version of a prion; prion is native to the animal, but the misfolding of it causes the pathology
The unfolded prion causes a domino effect to cause more and more native prion to unfold (causes SCRAPIE)
Scrapie results in formation of large B-amyloid fibrils (B-sheet structure) |
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Term
| Same ligand must be bound to both Fab sites on Ig - true or false? |
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Definition
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Term
| How many different Ig structures possible? |
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Definition
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Term
| What is the quaternary structure of Ig's? |
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Definition
They are HETEROTETRAMERS
2 identical light chains
2 identical heavy chains |
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Term
| How many domains make up 1 Ig molecule? |
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Definition
12 different Ig domains (Ig Folds)
Have 4/heavy chain, 2/light chain
ALL are unique (NOT identical) |
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Term
| What is the Ig fold/domain structure? |
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Definition
| 2 antiparallel B-sheets stacked on top of each other |
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Term
| What stabilizes the Ig fold? |
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Definition
Hydrophobic interactions between the antiparallel B-sheets
S-S bond connects sheets also |
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Term
| How many hypervariable loops are there? |
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Definition
3 on each V(l) and V(h)
12 in total |
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Term
| Which region of the Ig are the hypervariable loops present on? |
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Definition
| The VARIABLE region (present on all 4 variable domains, 2 light and 2 heavy) |
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Term
| How many hypervariable loops are there per Fab? |
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Definition
6
2 Fab in total, therefore 12 hypervariable loops |
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Term
| How is antibody-antigen binding accomplished? |
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Definition
By the INDUCED FIT MODEL
Loops can slightly change structure upon antigen binding (regions are dynamic)
Loops are NOT part of normal secondary structure |
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Term
| Where does the variability in the Fab of the antibodies stem from? |
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Definition
| The hypervariable loops regions - different primary structure (because primary dictates tertiary), gives rise to different loop regions |
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