Hemoglobin Disorders. Molecular Methods and Protocols

289 Pages·2010·2.653 MB·English

by  Ronald L. Nagel

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X-ray Crystallography of Hemoglobins 1
1
X-ray Crystallography of Hemoglobins
Martin K. Safo and Donald J. Abraham
1. Introduction
X-ray crystallography has played a key role in understanding the relation-
ship between protein structure and physiological function. In particular, X-ray
analysis of hemoglobin (Hb) crystals has been pivotal in the formulation of
basic theories concerning the behavior of allosteric proteins. Methemoglobin
(MetHb) from horse was the first three-dimensional (3D) structure of liganded
Hb to be solved (1–4). It was followed by crystallographic determination of the
unliganded (deoxygenated) form nearly a decade later (5). The X-ray analyses
provided 3D atomic resolution structures and confirmed that Hb was tetrameric,
containing two subunit types (α and β), and one oxygen-binding heme group
per subunit. John Kendrew (myoglobin) and Max Perutz (Hb) received the
Nobel Prize for their pioneering work, being the first to determine the 3D struc-
tures of proteins, using X-ray crystallography. Since the crystallographic deter-
mination of these structures, there has been an almost exponential increase in
the use of X-ray crystallography to determine the 3D structures of proteins,
i.e., as evidenced by the history of structures deposited in the protein data bank.
Comparison of the quaternary structures of liganded and deoxygenated horse
Hb clearly showed significantly different conformational states. The Hb X-ray
structures were the first to confirm the two-state allosteric theory put forward
by Monod et al. (6), which is referred to as the MWC model. The liganded Hb
conformation conformed to the MWC relaxed (R) state, while unliganded Hb
conformation conformed to the MWC tense (T) state. The source of the tension
in the T state was attributed to crosslinking salt bridges and hydrogen bonds
between the subunits. The relaxed (R) state has only a few intersubunit hydro-
gen bonds and salt bridges.
From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols
Edited by: Ronald L. Nagel © Humana Press Inc., Totowa, NJ
1
2 Safo and Abraham
Muirhead and Greer (7) published the first structure of human adult deoxy-
genated hemoglobin (deoxyHbA). Several years later, Baldwin and Chothia
(8,9) and Baldwin (9) published the structure of human adult carbonmonoxy-
hemoglobin (HbCOA), and Shaanan (10) published the structure of human
adult oxyhemoglobin (oxyHbA). Interestingly, the structure of oxyHbA was
delayed because of complications resulting from heme iron autoxidation. Sub-
sequently, a new quaternary ligand-bound Hb structure known as R2 (11) or Y
(12,13) provided another relaxed structure. R2 was proposed to be a low-energy
intermediate in the T-to-R allosteric transition. However, further analysis has
revealed that R2 is not an intermediate but, rather, another relaxed end-state
structure (14). Quite recently, our laboratory discovered two more novel HbCO
A relaxed structures (R3 and RR2); RR2 has a structural conformation between
that of R and R2 (unpublished results). The quaternary structural difference
between T and R3 is as large as that of T and R2. However, R2 and R3 have
very different conformations. The quaternary difference is determined by
superimposing the α1β1 subunit interfaces and calculating the rotation angle
between the nonsuperimposed α2β2 dimers (8,9).
The first 3D structures of horse Hb were solved using isomorphous replace-
ment techniques (1–3,5). A number of published Hb structures also crystallize
isomorphously, thus making it possible to use phases from the known isomor-
phous Hb structure for further structural analysis. The development of molecu-
lar replacement methods (15,16) for the solution of protein structures enabled
routine structure solutions for nonisomorphous Hb crystals.
When the structure horse Hb was determined, no computer refinement pro-
grams existed. Therefore, the atomic positions were refined visually against the
electron density map. With isomorphous mutant crystals (17) or isomorphous
crystals with bound allosteric effectors (18), simple electron density difference
map calculations have been shown to be powerful tools in analyzing structural
differences. Currently, all new protein structures are refined using modern, faster
computing methods, such as CNS (19) and REFMAC (20).
The crystal structures of more than 250 Hbs have been solved and published,
including mutants and Hb cocrystalized with allosteric effector molecules.
Selected examples of native and mutant Hbs including quaternary states, crys-
tallization conditions, and unit cell descriptions are given in Tables 1–3. The
structures of mutant Hbs provided the first concrete correlation between struc-
tural changes and disease states, while Hb cocrystallized with small effector
molecules has advanced our understanding of the fundamental atomic-level
interactions that regulate allosteric function of an important protein.
The general methodologies for isolating, purifying, crystallizing and crystal
mounting for data collection follow. The X-ray structure solution of Hb and
variants is routine and employs the techniques discussed above: isomorphous
Table 1
X
Crystallization Conditions and Structural Properties of Selected Human Hbs -r
a
y
Quater- Chemical Resolution
C
Name nary state form Crystallization condition Unit cell characteristicsa (Å) Referencer
y
s
DeoxyHbA T Normal 2.2–2.8 M NH4 phosph/sulfate, a = 63.2, b = 83.5, c = 53.8 Å, 1.7 25 ta
pH 6.5 β = 99.3°, SG = P21, AU = 1 tetramer llo
DeoxyHbA T Normal 10–10.5% PEG 6000, 100 mM a = 97.1, b = 99.3, c = 66.1 Å, 2.15 26 g
r
KCl, 10 mM K phosph, pH 7.0 SG = P21212, AU = 1 tetramer ap
RSR13-deoxy T Normal 2.5–2.9 M NH phosph/sulfate, pH 6.5 a = 63.2, b = 83.6, c = 53.9 Å, 1.85 27 h
4 y
HbA complexb β = 99.2°, SG = P21, AU = 1 tetramer o
DeoxyHbFc T Fetal 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 SG = P21, AU = 1 tetramer 2.5 28 f H
OxyHbA R Normal 2.25–2.75 M Na/K phosph, pH 6.7 a = 53.7, b = 53.7, c = 193.0 Å, 2.1 10 e
m
SG = P4 2 2, AU = 1 dimer
1 1 o
HbCO A R Normal 2.25–2.75 M Na/K phosph, pH 6.7 a = 53.7, b = 53.7, c = 193.8 Å, 2.7 8 g
SG = P4 2 2, AU = 1 dimer lo
3 1 1 b
HbCO A R2 Normal 16% PEG 6000, 100 mM, a = 97.5, b = 101.7, c = 61.1 Å, 1.7 11 in
s
Na cacodylate, pH 5.8 SG = P2 2 2 , AU = 1 tetramer
1 1 1
CO Gower II R2 Embryonic 21% MME PEG 5000, a = 62.8, b = 62.8, c = 320.9 Å, 2.9 29
(α2ε2)d 0.2 M TAPS-KOH, pH 8.5 SG = P4 2 2, AU = 1 tetramer
3 1
HbCO Ae R3 Normal 2.34–2.66 M Na/K phosph, pH 6.4–6.7 a = 61.5, b = 61.5, c = 176.3 Å, 2.65 Unpublished
SG = P4 22, AU = 1 dimer data
1
HbCO Ae RR2 Normal 2.34–2.66 M Na/K phosph, pH 6.4–6.7 a = 65.5, b = 154.6, c = 55.3 Å, 2.18 Unpublished
SG = P2 2 2, AU = 1 tetramer data
1 1
CNMetHbAf Y Normal 16–17% PEG 8000, 0.1 M Tris, a = 106.1, b = 86.2, c = 64.3 Å, 2.09 13
0.12% BOG SG = P2 2 2 , AU = 1 tetramer
1 1 1
a SG , space group; AU, and asymmetric unit.
b RSR13 is an allosteric effector.
c The authors of deoxyHbF did not provide the cell constants, however, the crystal is isomorphous to the high-salt deoxyHbA crystal (25).
d The quaternary structure of carbonmonoxy embryonic Gower II Hb lies between that of R and R2 states, though closer to the R2 state.
e Relaxed end-state structures (see text).
f The quaternary structures of Y and R2 state Hbs are similar. 3
4
Table 2
Crystallization Conditions and Structural Properties of Selected Natural Mutant Human Hbs
Quater- Chemical Resolution
Name nary state form Crystallization condition Unit cell characteristicsa (Å) Reference
DeoxyHbA
Sickle cell T Glu6βVal 33% PEG 8000, 5.5 mM citrate, pH 4.0–5.0 a = 52.9, b = 185.7, c = 63.3 Å, 2.05 24
β = 92.6°, SG = P2 , AU = 2 tetramers
1
Catonsville T Pro37α-Glu- 2.2–2.8 M NH Phosph/sulfate pH 6.5 a = 63.2, b = 83.6, c = 53.8 Å, 1.7 30
4
Thr38α β = 99.4°, SG = P2 , AU = 1 tetramer
1
Rothschild T Trp37βArg 10–10.5% PEG 6000, 100 mM KCl, a = 97.1, b = 99.3, c = 66.1 Å 2.0 26
10 mM K phosph, pH 7.0 SG = P2 2 2, AU = 1 tetramer
1 1
Thionville T Val1αGlu 2.2–2.8 M NH phosph/sulfate, pH 6.5 a = 63.2, b = 83.6, c = 53.8 Å, 1.5 31
4
AcetMet(-1)1α β = 99.4°, SG = P2 , AU = 1 tetramer
1
4 COYpsilanti Y Asp99βTyr 2.25–2.30 M Na/K phosph, pH 6.7 a = 93.1, b = 93.1, c = 144.6 Å 3.0 12
SG = P3 21, AU = 1 tetramer
2
Cowtown R His146βLeu 2.25–2.75 M Na/K phosph, pH 6.7 a = 54.38, b = 54.38, c = 195.53 Å, 2.3 32
SG = P4 2 2, AU = 1 dimer
1 1
Knossosb T Ala27βSer 2.2–2.8 M NH phosph/sulfate, pH 6.5 SG = P2 , AU = 1 tetramer 2.5 33
4 1
Grange- T Ala27βVal 2.2–2.8 M NH phosph/sulfate, pH 6.5 SG = P2 , AU = 1 tetramer 2.5 33
4 1
Blancheb
Brocktonb T Ala138βPro 2.2–2.8 M NH phosph/sulfate, pH 6.8 SG = P2 , AU = 1 tetramer 3.0 34
4 1
Suresnesb T Arg141αHis 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 SG = P21, AU = 1 tetramer 3.5 35 S
Kansas T Asn102βThr 2.2–2.8 M NH phosph/sulfate, pH 6.5 a = 63.4, b = 83.6, c = 53.9 Å, 3.4 36 a
4 β = 99.3 o, SG = P2 , AU = 1 tetramer fo
1
a
a SG, space group and; AU, asymmetric unit. n
d
b The authors of Hb Knossos, Grange-Blanche, Brockton, and Suresnes did not provide the cell constants, however, the crystals are isomorpho us
A
to the high-salt deoxyHbA crystal (25). b
r
a
h
a
m
X
-
r
a
y

Table 3 C
r
Crystallization Conditions and Structural Properties of Selected Artificial Mutant Human Hbs y
s
t
a
Quater- Chemical Resolution
Name nary state form Crystallization condition Unit cell characteristicsa (Å) Referenlloce
g
r
Yα42H T Tyr42αHis 2.2–2.8 M NH phosph/sulfate, pH 6.5 a = 62.4, b = 81.2, c = 53.3 Å, 1.8 38 a
4 p
β = 99.65°, SG = P2 , AU = 1 tetramer h
1 y
rHb(α96Val→Trp) T Val96αTrp 2.2–2.8 M NH phosph/sulfate, pH 6.5 a = 63.3, b = 83.4, c = 53.8 Å, 1.9 39
4 o
β = 99.5°, SG = P21, AU = 1 tetramer f H
rHb(α96Val→Trp) R Val96αTrp 2.25–2.75 M Na/K phosph, pH 6.7 a = 54.3, b = 54.3, c = 194.1 Å 2.5 39 e
m
SG = P4 2 2, AU = 1 dimer
Deoxy-Hbβ6W T Glu6βTrp 4–7 uL of 33 % PEG 8000, 5 uL a = 62.9,1 b1 = 81.3, c = 111.4 Å 2.0 40 og
of Na citrate, pH 4.8 SG = P2 2 2 , AU = 1 tetramer lo
5 Deoxy-rHb1.1 T Asn108βLys 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 a = 62.9,1 b1 =1 82.0, c = 53.9 Å, 2.0 41 bin
α1-Gly-α2 β = 99.0°, SG = P2 , AU = 1 tetramer s
1
CNmet-rHb1.1 B Asn108βLys 13 % PEG 3350, 10 mM KCN, a = 102.5, b = 115.2, c = 56.7 Å 2.6 41
α1-Gly-α2 150 mM NH acetate, pH 5.0 SG = P2 2 2 , AU = 1 tetramer
4 1 1 1
Deoxy-βV67T T Val67βThr 2.2–2.8 M NH phosph/sulfate, pH 6.5 a = 63.5, b = 83.2, c = 54.0 Å, 2.2 42
4
β = 99.15°, SG = P2 , AU = 1 tetramer
1
Des-Arg141αHbA T des-Arg141α 10–10.5 % PEG 6000, 100 mM a = 96.7, b = 98.7, c = 66.0 Å, 2.1 43
KCl, 10 mM K phosph, pH 7.0 SG = P2 2 2, AU = 1 tetramer
1 1
Bulltown T His146βGln 2.2–2.8 M NH phosph/sulfate, pH 6.5 a = 63.2, b = 83.4, c = 53.8 Å, 2.6 44
4
β = 99.4°, SG = P2 , AU = 1 tetramer
1
Deoxy-βV1M T Val1βMet 2.2–2.8 M NH phosph/sulfate, pH 6.5 a = 63.2, b = 83.7, c = 53.8 Å, 1.8 45
4
β = 99.4°, SG = P2 , AU = 1 tetramer
1
a SG, space group; AU, asymmetric unit.
5
6 Safo and Abraham
replacement, difference electron density calculations, molecular replacement,
and structure refinement (for details, see references).
2. Materials
2.1. Purification of Human Hb for Crystallization
1. HbA is purified from outdated human red blood cells (RBCs) unsuitable for trans-
fusion (~500 mL). Sickle cell Hb (HbS) is purified from sickle cell blood, nor-
mally obtained from homozygote sickle cell patients who receive blood-exchange
transfusions. To avoid clotting, blood samples are normally stored with about
1/10 vol of an anticoagulant agent, such as EDTA, heparin, or potassium citrate.
2. Buffer stock solution (5–10 L) containing 50 mM Tris buffer (pH 8.6) with
EDTA: The solution is made by mixing 50 vol of 0.1 M Trizma base, 12.4 vol of
0.1 N Trizma hydrochloride, adjusting the volume to 100 mL with deionized
water containing 4 g of EDTA (see Note 1).
3. Stock saline solutions (3 and 1 L) of 0.9% (9 g/L) and 1.0% NaCl (10 g/L),
respectively.
4. DEAE sephacel and chromatography column equipment.
5. Cellulose dialysis tubes (Fisher Pittsburgh, PA).
6. Carbon monoxide gas cylinder (Matheson, Joliet, IL) (see Note 2).
7. NaCl, Na dithionite, and K HPO .
2 4
8. Three Erlenmeyers or side arm flasks (1 L).
2.2. Crystallization of Human Hb
1. Cyrstallization procedures will be described for deoxyHbA, deoxyHbS, and COHb
A. These methods are also applicable to other HBs. HbA and HbS isolated and
purified as described in Subheading 3.1.2. are used for all crystallization setups.
2.2.1. High-Salt Crystallization of T-State deoxyHbA
1. HbA solution (12 mL) (60 mg/mL or 6g%): Dilute the protein with deionized
water if necessary to obtain the above concentration.
2. 3.6 M precipitant solution (50 mL) (pH 6.5): This is made by mixing 8 vol of 4 M
(NH ) SO , 1.5 vol of 2 M (NH ) HPO , and 0.5 vol of 2 M (NH )H PO .
4 2 4 4 2 4 4 2 4
3. Deionized water (100 mL).
4. Ten 8-mL sterile interior vacutainer tubes (Becton Dickinson, Franklin
Lakes, NJ).
5. Stoppered glass jar (Aldrich, St. Louis, MO).
6. Parafilm.
7. Pipets and pipet tips (100 and 1000 mL).
8. Three 15- to 25-mL beakers or volumetric flasks.
9. Graduated cylinders (10- and 50-mL).
10. Mixture of FeSO (2 g) and Na citrate (1.5 g).
4
11. A few grains of Na dithionite.
12. Test tube rack.
X-ray Crystallography of Hemoglobins 7
2.2.2. High-Salt Crystallization of R-State HbCO A
1. HbA solution (12 mL) (40 mg/mL or 4g%) in a 50-mL round-bottomed flask
equipped with a stir bar and a greased stopcock adapter.
2. 3.4 M precipitant solution (40 mL) (pH 6.4): This is made by mixing 7 vol of 3.4 M
NaH PO and 5 vol of 3.4 M K HPO (see Note 3).
2 4 2 4
3. Deionized water (100 mL).
4. Toluene (50 µL).
5. Ten 8-mL sterile interior vacutainer tubes (Becton Dickinson).
6. Stoppered glass jar (Aldrich).
7. Pipets and pipet tips (100 and 1000 mL).
8. A few grains of Na dithionite.
9. Carbon monoxide gas cylinder (Matheson) (see Note 2) and nitrogen gas
cylinder.
10. Test tube rack.
11. Vacuum pump and rubber tubing.
2.2.3. Low-Salt Crystallization of T-State deoxyHbS
1. HbS solution (1.2 mL) (120 mg/mL or 12g%).
2. 50% (w/v) polyethylene glycol (PEG) 6000 (12 mL).
3. 0.2 M citrate buffer (1 mL), pH 4.0–5.0 (Hampton Research, Laguna Hills, CA).
4. Deionized water (10 mL).
5. Ten 3-mL sterile interior vacutainer tubes (Becton Dickinson).
6. Parafilm.
7. Stoppered glass jar (Aldrich).
8. Pipets and pipet tips (100 and 1000 mL).
9. Two 15- to 25-mL beakers.
10. A few grains of Na dithionite.
2.3. Crystal Preparation and Mounting
The methods described here are for deoxyHbA and COHb A, and are also
applicable to other Hb cystals.
2.3.1. Room Temperature Data Collection
1. Vacutainer tube containing T- or R-state crystals.
2. Capillary sealant, such as epoxy or paraffin wax or any wax with a low
melting point.
3. Disposable pipets and pipet rubber bulb.
4. Stainless steel blunt-end needles (Fisher).
5. Disposable syringes (3–5 mL) (Fisher Scientific).
6. Sterilized paper wicks (Hampton Research).
7. Thin-walled quartz or borosilicate capillaries (Charles Supper, Natick, MA),
ranging in size from 0.1 to 1.2 mm.
8. Soldering iron.
9. Sharp tweezer.
8 Safo and Abraham
2.3.2. Cryogenic Temperature Data Collection
2.3.2.1. T-STATE DEOXYHBA CRYSTAL
1. Vacutainer tube containing T-state crystals.
2. Glycerol (100 µL).
3. Small Dewar flask with liquid nitrogen.
4. Thin fiber loop with diameter slightly larger than longest crystal dimension
(Hampton Research).
5. Cryovial and cryovial tong (Hampton Research).
6. Disposable pipets and pipet rubber bulb.
7. Glass slides.
8. A few grains of Na Dithionite.
2.3.2.2. R-STATE COHB CRYSTAL
1. Vacutainer tube containing R-state crystals.
2. Cryoprotectant solution made by mixing 60 µL of mother liquor and 5–8 µL of
glycerol.
3. Thin fiber loop with diameter slightly larger than longest crystal dimension.
4. Disposable pipets and pipet rubber bulb.
5. Glass slides.
3. Methods
3.1. Purification of Human Hb for Crystallization
About 90% of RBC content is made up of Hb, and in healthy human adults,
HbA accounts for more than 90% of the human Hb protein, while other minor
components, such as fetal HbF (~1%) and hemoglobin HbA (2 to 3%), make
2
up the remainder. The method described here for isolating HbA and HbS from
blood or RBCs, and further purification by ion-exchange chromatography, is a
modified version of Perutz’s (21) protocol. This procedure, using appropriate
buffer eluents, has also been used to separate other variant forms of human Hb
and Hb from other species.
3.1.1. Purification of HbA
1. Place three Erlenmeyer or side-arm flasks in a walk-in refrigerator and chill to 4°C.
2. Centrifuge the RBCs at 600g for 20 min at 4°C.
3. Gently aspirate the supernatant solution (debris, plasma, and excess serum) from
the centrifuge bottles and discard.
4. Wash the RBCs three times with an excess volume of 0.9% NaCl, and then once
with 1.0% NaCl, each time centrifuging and discarding the supernatant solution.
5. Pool the RBCs into a chilled flask and lyse the cells by adding 1 to 2 vol of 50 mM
Tris buffer, pH 8.6 (containing EDTA) (see Note 4).
6. Allow the mixture to stand on ice for 30 min with occasional gentle stirring.
7. Centrifuge the Hb solution at 10,000g for 2 h at 4°C.
X-ray Crystallography of Hemoglobins 9
8. Pool the supernatant Hb solution, which is free of cell debris, into a chilled flask,
and slowly add NaCl (40–60 mg/mL of Hb solution) while stirring the solution.
9. Centrifuge the Hb solution at 10,000g for 1 to 2 h at 4°C to remove any remain-
ing cell stroma.
10. Pool the clear supernatant Hb solution into a chilled flask and discard the “syr-
upy” pellet.
11. Dialyze the Hb solution against 50 mM Tris buffer, pH 8.6 (containing EDTA),
at 4°C to remove NaCl or other low molecular weight impurities (see Note 5).
12. Further purify the dialyzed Hb by ion-exchange chromatography using DEAE
sephacel to separate the HbA from other Hb components (see Note 6):
a. Equilibrate the resin with 50 mM Tris buffer, pH 8.6.
b. Run the Hb solution through the column with 50 mM Tris buffer, pH 8.6
(containing EDTA), to allow the various Hb bands to separate. HbA (light
2
band color) elutes first, followed by HbA (dark band color). The HbA frac-
tions can be examined for purity by electrophoresis and only pure fractions
(dark band) pooled together.
13. Concentrate the pooled fractions (40–100 mg/mL) with an Amicon stirred
cell (Model 402) to a final HbA concentration of about 80–120 mg/mL (see
Note 7).
14. Store the concentrated HbA, which is essentially the oxygenated form, at –80°C
or freeze in liquid nitrogen. Hb stored at this temperature can remain suitable for
crystal growth experiments for several years.
3.1.2. Purification of HbS
HbS from homozygous sickle cell blood is isolated and dialyzed as described
for HbA in Subheading 3.1.1. (steps 1–11). The HbS solution is further puri-
fied on a DEAE sephacel ion-exchange column using a buffer gradient of 50 mM
Tris buffer, pH 8.6 (containing EDTA), and 50 mM Tris buffer, pH 8.4 (con-
taining EDTA) (see Note 1).
1. Elute first HbA Tris buffer at pH 8.6, then HbS at pH 8.4.
2
2. Concentrate the pure HbS, identified by electrophoresis and store as indicated for
HbA in Subheading 3.1.1. (steps 13 and 14).
3.2. Crystallization of Human Hb
DeoxyHbA crystallizes from either high-salt or low-salt precipitants (7,21).
The ligand-bound R-state Hbs, such as oxyHbA, HbCO A, and MetHbA; gen-
erally crystallize under high-salt conditions (8–10,21), while the ligand-bound
R2- or Y-state HbAs also crystallize mainly under low-salt conditions (11,13).
The most common approach to crystallizing Hb is the Perutz’s (21) batch
method. Alternatively, the vapor diffusion method of hanging or sitting drop
(22) is used, especially when only a small amount of protein is available. Here,
detailed crystallization is described for both T- and R-state human HbA and