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BIOC 3560 > Regulation and Protein Function > Flashcards

Regulation and Protein Function Flashcards

1
Q

what properties of proteins interest us?

A
  • size (molecular weight)
  • shape (secondary structure, fold)
  • charge
    hydrophobicity/hydrophilicity
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2
Q

describe the reversible modulation of proteins

A

proteins are DYNAMIC MOLECULES
INFLUENCED by other molecules
can be STABLE or TRANSIENT

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3
Q

describe the STABLE interactions

A

a PROSTHETIC group is PERMANENTLY associated with a protein and required for functions

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4
Q

describe TRANSIENT interactions

A

a LIGAND is a molecule bound REVERSIBLY to a protein

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5
Q

what is a BINDING SITE?

A

region of a protein where the LIGAND binds

complementary to the ligand in size, shape, charge, hydrophobicity/hyrdophilicity

HIGHLY SPECIFIC
can have >1 binding sites

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6
Q

what is INDUCED FIT?

A

structural adaptations between protein and ligand

conformational change can make binding sites more complementary

ex. hexokinase and D-glucose

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7
Q

what are ENZYMES?

A

special case of protein-ligand interactions

substrate (~ligand): molecule CHANGED by enzyme

catalytic/active site: binds substrate and facilitates chemical transformations

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8
Q

what are the key concepts in protein-ligand interactions?

A

reversible-binding
specificity
conformational change
regulation

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9
Q

why do we need oxygen-binding proteins?

A

O2:
poorly dissolved in aqueous solution
inefficiently diffuses through tissues

large organisms need mechanisms for transporting O2
transporter must be specific and reversible

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10
Q

how are O2 molecules complexed?

A

with transition metals with a high affinity for O2 (ex. Fe, Cu)

Free iron can turn O2 into ROS –> tendency is reduced when Fe is in heme

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11
Q

what is HEME?

A

a PROSTHETIC GROUP

photopotphyrin IX + Fe2+

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12
Q

what are porphyrins?

A

four pyrrole rings
connected by methine (-CH=) bridges
linked into a conjugate C=C system
4 N atoms bind to metal ion in center
X groups (propionate in heme) define the type

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13
Q

describe the binding of Fe ions to O2

A

Fe2+ binds reversibly
Fe3+ does not bind

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14
Q

how is the oxidation of Fe2+ prevented?

A

Fe is buried within the molecule
one coordination bond is occupied by a side chain His residue
O2 binds reversibly at remaining position

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15
Q

what is myoglobin

A

monomer
binds and stores O2 in muscle

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16
Q

what is hemoglobin

A

tetramer (made of 2 a-globins and 2 B-globins)
O2 transporter

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17
Q

what is leghemoglobin

A

found in leguminous plants
sequesters O2, protecting O2 sensitive enzymes in N fixing plants

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18
Q

what is the structure of myoglobin?

A

single chain
153 residues
16.7 kDa

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19
Q

what are the advantages of the Hb/Mb naming system?

A

residues are different between globins, help us identify differences

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20
Q

why isn’t the naming system used in other proteins?

A

globins are well studied with X-ray crystallography
globins only have a-helices, would get convoluted with B-sheets
allows us to compare within a family

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21
Q

describe the globin structure naming system

A

globins are GLOBULAR a-helical proteins
8 a-helices are denoted A-H (N- to C- terminal)
connecting loops are identified by the two helices they join (CD, DE, etc)
AAs are identified by their RELATIVE positions within the motif (F8 = 8th AA on helix F, CD1 = 1st AA in loop between C and D)

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22
Q

how do we name the N- and C- term in globins

A

no helix n, “N” DENOTES THE N-TERM
“C” DENOTES THE C TERM
anything HCX is the C-TERM (where X is a number)

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23
Q

describe mb’s HEME BINDING pocket

A

HEME BINDING pocket formed between E and F helices
propionate (COO-) group side chains if heme are NEAR SURFACE of globin
rest of heme is surrounded by NON POLAR residues

His = polar
Heme is flanked by 2 polar and many non-polar residues

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24
Q

what is the role of the two histidines?

A

one his residue (F8) is DIRECTLY BONDED to Fe2+ (5th coordination bond –> PROXIMAL HISTIDINE (F8)

E7 is CLOSE but NOT BONDED to heme –> DISTAL HISTIDINE (E7)

O2 binds to Fe2+ on the E7 side of the atom (6th COORDINATION BOND)

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25
describe the proximal histidine
a his 8 residue at F8 is DIRECTLY BONDED to Fe2+ his F8 forms a 5th COORDINATION BOND for Fe2+ his F8 is known as the PROXIMAL HISTIDINE
26
describe the distal histidine
second his (E7) is CLOSE TO but NOT BONDED to HEME (DISTAL HIS) O2 binds to to Fe2+on the E7 residue (6th COORDINATION BOND) this allows O2 to be bonded at an ANGLE --> favourable interaction
27
how are linking sequences similar between different globins?
only 18% alignment CONSERVED b/w globins PROXIMAL AND DISTAL his residues are CONSERVED IN ALL 3 globins
28
what does O2 binding depend on in Mb?
STRUCTURE around ligand binding site --> DISTAL and PROXIMAL HIS FLEXIBILITY of protein (dynamic) "BREATHING": small (<1A) molecular motions of AA side chains on a nanosecond scale (allows O2 atom to wiggle into molecule)
29
how is O2 binding measured?
CONJUGATED BOND systems causes a STRONG ABSORBTION of VISIBLE LIGHT O2 binding affects the ELECTRON DISTRIBUTION an d alters ABSORBTION OF LIGHT by heme OXY and DEOXY forms of heme have different SPECTRA OXY: absorbs BLUE DEOXY: absorbs RED used experimentally to measure O2 binding
30
what are the variables in ligand binding?
[p] = concentration of FREE PROTEIN (not ligand bound) [L] = ligand concentration [PL] = concentration of ligand=bound protein Kd = DISSOCAITION constant = [L] when half the binding sites are occupied (measures STRENGTH of interactions) Ka = association constant (inverse of Kd)
31
what is the formula for kd?
Kd = [P][L]/[PL] = 1/Ka high Kd = weak binding
32
why are these terms important?
describe REVERSIBLE BINDING STRENGTH of binding (evolved to be strong) INHIBITION of binding (non-static, phosphorylation and post-transcriptional modifications can change protein)
33
what is the formula for occupancy?
Y = binding sites occupied / total binding sites Y = [PL] / [PL] + [P] thus, we can derive: Y = [L] / [L] + Kd
34
describe the shape of a ligand binding curve?
Y is a HYPERBOLIC function of [L] {y E 0,1} kd can be found at Y=0.5 [L] on the y-axis is not the same as [L] in the equation --> has to be in excess
35
what are some typical Kd ranges?
typical: x10^-10 - x10^-5 sequence specific: x10^-11 - x10^-5 antibody: x10^-12 - x10^-8 (HIGH AFFINITY = LOW KD) enzyme substrate: x10^-7 - x10^-3 (LOW AFFINITY = HIGH KD)
36
what formula do we use for O2 binding proteins?
Y = pO2 / pO2 + P50 pO2 = partial pressure of O2 (analogous to [L]) Y = occupancy P50 = partial pressure of O2 at which half the ligand binding sites are occupied
37
why is the protein conformation critical for the specificity of protein-ligand interactions?
CO binds to FREE heme with 20000x greater affinity than O2 CO binds to heme in Mb with only 200X the affinity of O2 DUE TO STERIC HINDERANCES beween His E7 and CO and a favourable H bond between O2 and His E7
38
what is the structural basis for specificity?
O2 binds to heme at an angle CO binds to heme UPRIGHT (bumps into DISTAL his) create STERIC HINDERANCES between distal His E7 and CO makes a FAVOURABLE BOND between O2 and E7
39
describe O2 binding by mb
binds to O2 with HIGH AFFINITY (only need a little bit of ligand to get to P50) for a wide range, mb is INSENSITIVE TO O2 (plateau) PO2 in tissues ~ 4kPa --> Mb is essentially SATURATED
40
compare the globin sequences of mb and hb
F4 is CONSERVED throughout F8 is CONSERVED throughout in different globins, segments of (mostly) different AAs form similar structures out of ~150 AAs ONLY 27 ARE IDENTICAL (18%) --> conserved ones are very important this number usually appears BY CHANCE between 2 random proteins
41
describe Hb
major CARRIER of O2 TETRAMER (4 chains, 1 heme each) contains 2 types of globins (2 a chains of 141 AA and 2 B chains of 146 AAs) strongest interactions occur between a1-B1 and a2-B2 interfaces interactions are COMPLEMENTARY between pairs (BY SYMMETRY)
42
what types of interactions hold subunits together?
HYDROPHOBIC --> get away from H2O Hydrogen bonds --> H w/ N or O ION PAIRS --> salt bridges same chemical principles as ligands 30 residues involved in a1-B1 and a2-B2 subunits 19 residues involve din interface between a2-B1 and a1-B2
43
what is pO2 in the lungs and peripheral tissues?
lungs = 13kPa tissues = 4kPa
44
how is Hb an O2 transporter?
Mb binds to O2 with HIGH AFFINITY --> not good at releasing, P50~0.26kPa Hb binds O2 efficiently in the lungs and releases it easily in peripheral tissues (apparent P50 = 3.5kPa)
45
describe the Hb transition
ALLOSTERY subunits ROTATE towards each other Hb can undergo conformational changes from a low affinity (T) state to a high affinity (R) state
46
describe Hb allostery
NOT specialized O2 binding to one subunit of Hb ALTERS the affinity for O2 in adjacent subunits Induced conformational changes push adjacent subunits into the R state --> then bind to O2 with greater affinity cooperative binding: ALLOSTERIC EFFECT (one sight effects another at a distance)
47
what are allosteric proteins?
BINDING of a ligand at ONE SITE AFFECTS binding properties at ANOTHER
48
what are homotrophic allosteric modulators?
ligand and modulator are IDENTICAL ligand is essentially the modulator
49
what are heterotrophic allosteric modulators?
ligand and modulator are DIFFERENT can be ACTIVATORS (+) or INHIBITORS (-) for Hb: O2 is a ligand and an ACTIVATING homotrophic modulator
50
describe the difference in heme in the T and R state
T state is PUCKERED
51
describe the binding of O2
causes repositioning of the Fe2+ within heme, reducing the "pucker" of the porphyrin ring The change in the shape of heme pulls the proximal histidine along causing a shift in the position of helix F one of the adjustments that triggers the R transition, including BREAKING THE ION PAIRS by His HC3
52
what is deoxy Hb?
T state ION PAIR between His HC3 of the B subunit and both Asp FG1 of the B subunit and Lys C5 of the a subunit salt bridges exist when O2 unbound
53
describe the differences in His between the T and R states
R state (OXY) --> His HC3 is NOT PARTICIPATING in ion pairs that stabilize the T-state T-->R state transition is induced by O2 binding to Hb T = TENSE salt bridges R = RELAXED salt bridges (still has them)
54
describe O2 transport with a sigmoidal curve
lungs: HIGH pO2 (binds a lot of O2) tissues: low pO2 (Hb RELEASES a lot of O2) change in Y reflects how much O2 is released in tissues (38% of Hb's total capacity)
55
what is the Kd equation for n ligand binding sites?
Kd = [P][L]^n/[PLn]
56
what is the hill equation?
log(Y/1-Y) = nlog[L] - log Kd *assumes all sites log(Y/1-Y) = nlogpO2 - nlogP50
57
what is the difference between n and nH?
n = # of binding sites nH = Hill coefficient
58
what are the ranges of hill coefficients?
< 1 = negative cooperativity >1 = positive cooperativity = 1 = binding is not cooperative
59
what is the CONCERTED model?
subunits are FUNCTIONALLY IDENTICAL can exist in >1 conformations (generally 2 states (ex. T and R) all subunits CHANGE CONFORMATION SIMULTANEOUSLY more ligand added = HIGHER AFFINITY
60
what is the SEQUENTIAL model?
accurate, hard to understand ANY COMBINATION ligand binding can induce conformational change in subunits INDEPENDENTLY (spend more time in high affinity)
61
describe the changes in structure (T-->R) on binding
shape is critical to determine ALOSTERY SIGMOIDAL O2-binding curve of Hb is diagnostic of cooperative binding
62
what does cooperative binding require?
>1 ligand binding site interactions between ligand binding sites --> something that induces a change in another
63
what is worse: anemia vs 50% CO poisioning?
CO --> minimal amounts of O2 released
64
decribe the transport of O2 and CO2 by Hb
INVERSE RELAAtIONSHIP between the binding of O2 s binding of H+ and/or CO2 NEGATIVE heterotrophic ALLOSTERY EFFECT of pH and CO2 is BOHR effect
65
decribe the transport of O2 and CO2 by Hb in tissues
pH is lower --> [H+] and [CO2] is high Hb binds H+ and CO2 (DECREASED AFFINITY FOR O2) O2 RELEASED
66
decribe the transport of O2 and CO2 by Hb in the lungs
CO2 excreted pH rises Hb releases H+ and CO, increasing affinity for O2
67
describe Hb binding of H+
pH is very tightly reguated H+ binds several side chains in Hb, such as His HC3 protonation of His HC3 favours formation of the ion pair with Asp FG1 STABILIZES Hb in the T-state as pH drops, adoption of T-state encourages O2 to be released
68
describe Hb binding of CO2
reacts with a-amino groups at the N-term of each globin chain product is carbamionhemoglobin, containing a carbomate group carbomate groups participate in SALT BRIDGES that stabilize the T-state rxn produces H+ contributing to bohr effect
69
how does BPG regulate 2,3-bisphosphoglyerate
BPG binds in the centeral cavity of Hb forms a SALT BRIDGE w/ the two B subunits BPG stabilizes the T-state (deoxy Hb) (RELEASE of O2) REDUCES AFFINITY of Hb for O2 HETEROTROPHIC ALLOSTERIC modulator
70
what happens to the BPG binding site in the R-state?
it CLOSES UP
71
describe how BPG binding alternates with O2 binding
BPG binds to the T-state (DEOXY) ONLY binding O2 pushes Hb into the R state --> FORCES RELEASE of BPG BPG binding in lower O2 environments help push Hb to RELEASE O2 Hb in erythrocytes w/o BPG would barely release O2 in tissues
72
what is the role of BPG in fetal blood
HbF = a2Y2 HbF has a LOWER AFFINITY for BPG (Kd for BPG higher than HbA) HbF has a HIGHER AFFINITY FOR O2

BIOC 3560 (2 decks)

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