11
ANNOUNCEMENTS:
LECTURE READING & PROBLEMS AS IN LG FOR THIS WEEK; PyMOL #4, HEMOGLOBIN
QUIZZES THAT HAVE BEEN REGRADED AND RECORDED ARE IN THE GREEN FOLDER;
EACH FRIDAY, THESE WILL BE BROUGHT OVER TO THE BIOLOGY CENTER
FRIDAY'S LECTURE:
ALL INFO ABOUT PROTEIN FOLDING IS IN THE AA SEQUENCE. BUT CORRECT
DISULFIDES, AND CORRECT cis vs trans NEXT TO PROLINE ARE SLOW WITHOUT
ENZYMES.
PROTEINS CAN FOLD IN A SHORT TIME, TYPICALLY SEC - MIN: NOT UNDERSTOOD!
DETAILS OF FOLDING PROCESS? SEC STRUCTURE FORMS FIRST, OR "HYDROPHOBIC
COLLAPSE" FIRST? THE "MOLTEN GLOBULE" STATE FORMS BEFORE NATIVE FOLD
MISFOLDING; ROLES OF MOLECULAR CHAPERONES
** SOME DISEASES ARE CAUSED BY PROTEIN MISFOLDING: "AMYLOID DEPOSITS" AR
AGGREGATES OF MISFOLDED PROTEIN. HUNTINGTON'S DISEASE, ALZEIMER'S, THE
PRION DISEASES: PROTEINS
ALL
MOVEMENTS WITHIN ALL PROTEINS; EXAMPLES OF HIV PROTEASE, HIV CAPSID,
HEXOKINASE, AND Mb
TODAY:
PROTEIN FUNCTION CONNECTED TO PROTEIN STRUCTURE:
START OF THE HEMOGLOBIN STORY
TRANSPORT PROTEINS: THE O2 CARRIERS
ROLE OF HEME
Mb SHAPED TO FIT HEME, HOLD IN CREVICE
WHAT WOULD BE THE IDEAL PROPERTIES OF A MOLECULE DESIGNED TO
PICK UP O2 IN LUNGS, DELIVER O2 TO THE TISSUE?
WANT IT SATURATED WITH O2 IN LUNGS, BUT GIVING UP O2 IN TISSUE
1.0
OH NO, Mb STILL HOLDING ITS O2 IN TISSUE!!
Mb
=
Hb
FRACTION
OF HEMES
SATURATED
WITH O2
NOTICE THE SHAPE OF THIS
CURVE: "SIGMOID"
0.5
THIS MUCH O2 DROPPED
OFF TO TISSUE BY Hb
IN LUNGS
IN O2-STARVED TISSUE
00
25
50
O2 PRESSURE, mm Hg
75
100
Hb A 2 2
COMPARE Mb and Hb
TETRAMER
chain141 AA TETRAMER MW 65kD
chain 146 AA
Mb
N-TERMINUS OF Mb, or Hb or Hb
COMPARE III OF CHAINS OF Hb AND Mb:
3D: , , AND Mb ~ IDENTICAL; THIS PACKING OF 8 -HELICES
CALLED GLOBIN FOLD 8 HELICES LETTERED A-H
AA SEQ: AND 50% IDENTICAL, BOTH 75% DIFFERENT FROM
Mb
3D STRUCTURE
CONSERVED DURING 100s
OF MILLIONS OF YEARS
OF EVOLUTION, BUT AA
SEQUENCE VARIES SO
MUCH!
A SUBJECT OF ACTIVE
RESEARCH
JARGON! "CONSERVED"
SOME AA, KEY TO
EITHER STRUCTURE OR
FUNCTION, ARE
CONSERVED IN ALL 300
DIFFERENT Mb AND Hb
SEQUENCED SO FAR
Hb
Hb
Mb
Hb
Mb
THE SPECIAL PROPERTIES OF Hb
1. SATURATED WITH O2 IN LUNGS
2. GIVES UP O2 TO TISSUE
3. HIGHER O2 AFFINITY OF FETAL Hb OVER ADULT Hb
4. LOWER O2 AFFINITY AT LOWER pH
5. TRANSPORTS CO2
(UPPER CASE "K" FOR
EQUILIBRIUM CONSTANTS)
LOWER CASE "k" FOR
RATE CONSTANTS.
oxyHb
deoxyHb
kASSOC
DESCRIPTION OF O2 BINDING USING AN EQUATION: Mb + O2
kASSOC =
kDISSOC =
ASSOCIATION RATE CONSTANT, WHERE ASSOCIATION RATE =
kASSOC [Mb][O2]
DISSOCIATION RATE CONSTANT, WHERE DISSOCIATION RATE = kDISSOC [MbO2]
KEQ = [MbO2]/[Mb][O2] = kASSOC/kDISSOC
kDISSSOC
MbO2
BINDING OF O2 BY Hb --> THE SIGMOID CURVE
THE "SIGMOID" SHAPE DESCRIBES O2 BINDING
1.0
+ O2
+
O2
)3
O2
b(
H
2)
2
Hb
(O
O2 BINDING TO Hb is COOPERATIVE
2
O2's THAT ARE ALREADY BOUND
COOPERATE TO MAKE NEXT O2
BINDING MORE FAVORABLE
K2
O
+
K3 K4 300 x K1
IMPORTANT JARGON:
K3
0.5
K4
)4
Hb(O 2
)1
O2
(
Hb
0
Hb
+
O2
WITH pO2 = 0, WE
START WITH NO O2
BOUND, i.e. Hb
K1
50
O2-STARVED
TISSUE
pO2 , mmHg
100
APPROX
pO2 in
LUNGS
THE HILL EQUATION: SEPARATE COOPERATIVITY FROM PRESSURE AFFINITY
COOPERATIVITY
O2 FOR 50% BINDING, i.e. = 0.5
SMALLER P50 MEANS HIGHER O2 AFFINITY
log(
log( /1- ) = n log(pO ) - logP
2
50
HILL COEFFICIENT. HIGHER n
MORE COOPERATIVE
n MEANS: HOW MANY O2 COOPERATE TO BIND "ALL AT ONCE"
For Hb, n = 2.8, P = 26 mmHg
Hb HAS LESS O2 AFFINITY THAN Mb.
(HIGHER O2 PRESSURE REQUIRED
50
pO2
P50
TO GET = 0.5)
BUT ISOLATED OR
CHAINS ARE LIKE Mb!
For Mb, n = 1.0, P = 1 mmHg
50
pO2
P50
SOMETHING ABOUT Hb IV STRUCTURE
MAKES O2 BINDING LESS FAVORABLE
oxyHb
deoxyHb
THE IV STRUCTURES LOOK SO SIMILAR, BUT
THE HILL EQUATION, CONT'D
O2
O2
AND ENDS UP,
WITH 4 O2 BOUND,
LIKE THIS
oxy
pO2
Hb STARTS OFF
LIKE THIS
deoxy
WHY COOPERATIVITY? A
CLUE FROM THE CRYSTALS
deoxyHb CRYSTAL
O2
3D STRUCTURE OF EACH SUBUNIT (X-RAY,
NMR STUDIES) IS ~ SAME FOR OXY AND
DEOXY FORMS OF Hb
BUT THE IV OF THE O2-BOUND Hb DIFFERS
FROM THE IV OF THE DEOXY Hb THE
CRYSTAL SHATTERS
TWO DIFFERENT FORMS OF Hb!
SOMETHING ABOUT Hb IV STRUCTURE
CHANGES O2 BINDING STRENGTH
CRYSTAL
SHATTERS!
A CLOSE LOOK: SALT BRIDGES OF DEOXY-Hb NOT FOUND IN OXY-Hb
HEME
DISTORTED DIAGRAM OF SALT BRIDGES IN DEOXY-Hb
126-141
2
BPG
1
HEME
2
HEME
ZOOM ASP126 - ARG141
-
+
1
THESE 8 AA PAIRS CLOSE IN DEOXY, NOT IN OXYHb, SO DEOXY HAS POTENTIAL EXTRA
STABILIZATION FROM THESE EXTRA ION PAIRS.
THESE 8 ION PAIRS ALL BROKEN IN OXY-Hb
+
ION PAIR EXISTS
IN deoxyHb
-
FIRST O2 STRAINS OR BREAKS SOME ION
PAIRS IN ORDER TO BIND, SO BINDING IS WEAK;
NEXT ~ 3O2 BIND WITHOUT NEEDING TO BREAK
ION
SO BINDING IS HIGHER AFFINITY
O2 BPAIRS, ON ONE HEME GREATLY INFLUENCES
OUND
+
-
RE-STATE COOPERATIVE O2 BINDING:
NO ION PAIR
IN oxyHb
O2 BINDING ON DISTANT HEMES
JARGON: WHEN ONE PART OF A PROTEIN
INFLUENCES A DISTANT PART OF SAME PROTEIN,
EFFECT CALLED ALLOSTERIC = OTHER PLACE
BUT 8 NOT ENOUGH: DEOXY-Hb IS STABILIZED BY ADDITIONAL ION PAIRS:
BPG
MAMMALS; ALWAYS IN RBC; Hb/BPG = 1/1
DEOXY-Hb
BIRDS
FISH
FROM DEOXY
OXY
SHIFTING AND ROTATION
OF SUBUNITS CLOSES
CAVITY FOR BPG.
OXY-Hb CANNOT BIND BPG
Hb WITHOUT BPG
+
APPROX. 5 ION
PAIRS TO BPG
Hb + BPG: THIS IS WHAT
WE MEAN BY Hb!!
+ -+ - +
+ - BPG -+ +
+
WHY WOULD O2 BINDING DISRUPT DISTANT ION PAIRS?!
i.e. HOW IS HEME BINDING OF O2 CONNECTED TO SUBUNIT
CONTACTS OF IV?
(A CHANGE IN IV IS A CHANGE IN SUBUNIT CONTACTS!)
F-HELIX
EXPLANATION PROPOSED BY MAX PERUTZ, NOBEL PRIZEWINNER WHO GREATLY INFLUENCED UNDERSTANDING OF Hb:
~ 0.5
Fe
PROXIMAL HIS IS BOUND TO Fe2+,
MOVES WHEN O2 BINDS,
DRAGGING HELIX F, BREAKING
SUBUNIT ION PAIRS
O2
MORE GENERAL PICTURE OF WHY IV
STRUCTURE CAN LEAD TO MODIFIED
ACTION (ALLOSTERY, COOPERATIVITY):
JARGON ALERT! IV HAS TWO STATES
R-STATE
T-STATE
RELAXED
TENSE
Fe
STRONG O2
BINDING
WEAK O2
BINDING
TINY CHANGE IN Fe2+ POSITION LEADS TO BIG CHANGE IN IV
LET'S SEE WHY COMPARISON TO SIMPLE PROTEIN, Mb
QUIZ 3
KNOW
DON'T KNOW
LG pp. 61-85
LG problems 10-17
IV structure
recognize specific
folds
Last year's Quiz 3
Protein stability: G = H - T S
and be sure to get the signs right!
diagrams of Go vs structural coordinates
specificity of binding/
interaction domains
PyMOL assignment #3: Protein Folds,
including the Folds Supplement file
BRING A CALCULATOR but NOT A GRAPHING CALCULATOR