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THE LOCAL ELECTROSTATIC ENVIRONMENT DETERMINES CYSTEINE
REACTIVITY OF TUBULIN
P. J . BRITTO, LESLIE KNIPLING AND J. WOLFF*
Laboratory of Biochemistry and Genet ics
NIDDK, NIH, Bethesda, MD 20892
Running Ti t le: Tubul in Cysteine React iv i ty
*Corresponding author. E mai l : [email protected]
Tel . : 301 496 2685
Fax: 301 402 0240
JBC Papers in Press. Published on May 21, 2002 as Manuscript M204263200 by guest on A
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SUMMARY
Of the 20 cysteines of rat brain tubul in some react rapidly wi th sul fhydryl
reagents and some slowly. The fast-react ing cysteines cannot be dist inguished
wi th [1 4C]- iodoacetamide, [ 1 4C]-N-ethylmaleimide or IAEDANS, s ince modi f icat ion
to mole rat ios <<1 cysteine per d imer always leads to label ing of 6-7 cysteine
residues. These have been ident i f ied as C305:α , C315:α , C316:α , C347:α ,
C376:α , C241:β1 and C356:β1 by mass spectroscopy and sequencing. This lack
of speci f ic i ty can be ascr ibed to reagents that are too react ive; only wi th the
relat ively inact ive chloroacetamide could we ident i fy C347:α as the most react ive
cysteine of tubul in. Using the 3.5Å electron di f f ract ion structure i t could be
shown that the react ive cysteines were wi th in 6.5Å of posi t ively charged
argin ines and lys ines or the posi t ive edges of aromat ic r ings, presumably
promot ing dissociat ion of the th io l to the th io late anion. By the same reasoning
the inact iv i ty of a number of less react ive cysteines could be ascr ibed to
inhibi t ion of modi f icat ion by negat ively charged local environments, even wi th
some surface–exposed cysteines. We conclude that the local e lectrostat ic
environment of cysteine is an important , though not necessar i ly the only,
determinant of i ts react iv i ty .
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INTRODUCTION
The 20 SH groups of the tubul in d imer have long led to speculat ion as to
their funct ion. ‘Requirements’ for a few of the SH groups have been ident i f ied.
Thus, C12:β is near the binding s i te of the exchangeable GTP of β - tubul in (1) , and a
C12S:β mutat ion is lethal in haploid yeast a l though a C12A:β mutat ion is surv ivable
(2) . C241:β1 and C356:β1 are near, or are part of , the binding s i te for colchic ine
and other agents (3-5) . What the precise ro le of the ‘ involvement ’ of these cysteines
may be is, for the most part , not c lear. Some of the SH groups of tubul in form
thioesters wi th palmit ic acid both in v ivo and in v i t ro; these may be responsible for
membrane local izat ion of tubul in (6-9). One of these has been located as C376:α
(10). Except for th is palmitoylat ion s i te, no speci f ic funct ions have been ident i f ied
for the 12 SH groups of α - tubul in, and the order of react iv i t ies of the SH groups has
not been def in i t ively establ ished. I t has been repeatedly demonstrated (11) that
react ion of an equivalent of 1 or 2 SH groups wi th the usual a lkylat ing agents
abol ished polymerizat ion competence but their locat ion in the sequence has not
been unambiguously determined. Loss of colchic ine binding requires modi f icat ion of
addi t ional SH groups by these non-speci f ic SH reagents. For th is reason we have
approached the react iv i ty of tubul in SH groups in a more general sense, compar ing
the ef fects of th ioether, d isul f ide and th ioester format ion as wel l as their locat ion in
α - tubul in and β - tubul in.
Protein sul fhydryl groups can be involved in numerous react ions such as
oxidat ion, d isul f ide interchange, th ioether and th ioester format ion. For the
purpose of th is d iscussion, we shal l exclude oxidat ion. Al though f ree radical
react ions of SH groups are known, the remaining react ions al l involve the th io late
anion as the react ive species whereas the th io l group has very much lower
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react iv i ty (12). Cysteine react iv i ty toward var ious sul fhydryl reagents is regulated
by a number of factors including:
1 . Exposure to the solvent.
2. Dissociat ion of the th io l to the th io late anion. RS- is a strong nucleophi le
(st ronger than RO-) normal ly leading to SN2 react ions. Ionizat ion is suppressed by
neighbor ing acid ic groups and enhanced by bas ic amino acids (12). Whi le the
great preponderance of SH groups involved catalyt ical ly in enzyme react ions have
low pKa values for d issociat ion to the th io late anion, less is known about pKa
values of SH groups of cysteines not d i rect ly involved in catalysis. The tubul in
cysteine pKa values are not known. In general , i t is assumed, and has been shown
in certa in cases, that these approach the ‘normal ’ SH pKa near pH 8.5-9.0.
3. The react iv i ty of the SH reagent. For d isul f ide interchange the pKa is
ary lSH<<alkylSH, making the former more react ive. Thus, DTNB2 is h ighly react ive
both wi th respect to rate and extent of react ion wi th nat ive tubul in. Factors
out l ined in 4. and 6. below also contr ibute to th is h igh react iv i ty of DTNB. I t must
be remembered that many of the SH reagents can also react wi th undissociated
th io ls, a lbei t at a much lower rate. This must be kept in mind when ascr ib ing low
pKa values for SH groups f rom react iv i ty wi th a th io l reagent.
4. Charge compat ib i l i ty between reagent and the cysteine environment, e.g.
iodoacetate vs. iodoacetamide or DTNB vs. 2,2’ -d ipyr idyl d isul f ide (13). The last
named yie lds s igni f icant react ion wi th cysteine at pH 2. Because the tubul in pKa
values are not known, we tested cysteine (assuming a pKa~8.5) react iv i ty at pH 2.0
and found a br isk react ion wi th th is reagent. Presumably th is indicates that the
weakly nucleophi l ic , undissociated th io l was one of the reactants.
5. The stabi l i ty of the bonds formed ( in decreasing order) – th ioether> disul f ide>
th ioester. Most studies on tubul in SH groups have used th ioether format ion wi th
iodoacetate, iodoacetamide, their der ivat ives, or maleimides. The former react
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relat ively s lowly wi th nat ive protein and wi th a rather l imi ted number of cysteines
(11).
6. The nature of the leaving group of the sul fhydryl reagent – e.g. a th io late, as
found in DTNB, is a good leaving group, as are other negat ively charged species.
The great d i f f icul ty in analyzing very hydrophobic pept ides produced by
palmitoylat ion led us to take advantage of the greater stabi l i ty and hydrophi l ic i ty of
the th ioether bond for subsequent manipulat ions such as the analysis of t rypt ic
pept ides. In the present study we have focused on the comparat ive react iv i t ies for
th ioether format ion of the SH groups of tubul in. In a subsequent study we shal l
compare th is wi th d isul f ide and th ioester bond format ion, the ef fect of the loss of
the fast- or s lowly react ing cysteines, and the ef fect of the s ize of the subst i tuents
on the abi l i ty of tubul in to polymerize and to react wi th l igands.
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EXPERIMENTAL PROCEDURES
Mater ia ls- N-[ethyl-1-1 4C]ethylmaleimide (50 mCi/mmol) in n-pentane, [1-1 4C]-
iodoacetamide (50 mCi/mmol) in ethanol and chloroacetamide [carbonyl- 1 4C] (55
mCi/mmol) were purchased f rom Amer ican Radiolabeled Chemicals (St . Louis,
MO). N-Ethylmaleimide and iodoacetamide were f rom Sigma and 1,5-IAEDANS [5-
((( (2- iodoacety l )amino)ethyl)amino)2 naphthalene-1-sul fonic acid] was f rom
Molecular Probes. Syn -Monobromobimane ( f rom Molecular Probes) and Thioglo 1
( f rom Calbiochem) were used f rom their acetoni t r i le stock solut ions. Trypsin-TPCK2
was obtained f rom Worthington. Al l other reagents were the highest grade
avai lable f rom Sigma unless otherwise noted. Pure (>99%) rat brain tubul in was
prepared as descr ibed (14). Al l the exper iments were performed wi th the fo l lowing
buf fer in the dark: 0.3 M Mes (pH 6.9), 1.0 mM EGTA (ethyleneglycol-b is-(β amino-
ethyl ether)N,N’- tetraacet ic acid)2 and 1.0 mM MgCl2 , and tubul in concentrat ion
was 30 µM in al l exper iments. Stock solut ions of the sul fhydryl reagents were
prepared f resh in Mes a.b. (assembly buf fer) (0.1 M Mes, 1.0 mM EGTA, 1.0 mM
MgCl2, pH 6.9). The speci f ic act iv i t ies of 1 4C reagents were adjusted wi th
unlabeled compounds whenever necessary. The RP-HPLC columns were obtained
f rom Phenomenex.
Sulfhydryl Modi f icat ions wi th [ 1 4C]- Iodoacetamide, [ 1 4C]-N-Ethylmaleimide,
[1 4C]-Chloroacetamide and 1,5- IAEDANS - Two types of exper iments were
performed: (1) t ime course of tubul in sul fhydryl modi f icat ion at 37 °C at low
(1:2) and high (50:1) molar rat ios of reagent to tubul in, and (2) 8 h
incubat ion at 4°C with vary ing molar rat ios (1:5, 1:2, 1:1, 3:1, 5:1 and 10:1)
of reagent to tubul in. React ions were stopped by adding β -mercaptoethanol
to a f inal concentrat ion of 5 mM, samples were sonicated and placed on ice.
The modi f ied tubul in was separated immediately f rom the unreacted reagent
and β -mercaptoethanol by passing through a Sephadex G25-Medium column
(25 cm x 0.7 cm) equi l ibrated wi th 10 mM Tr isHCl buf fer (pH 8.5) . The
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protein f ract ions were pooled and protein was est imated using bic inchoninic
acid assay (15). The radioact iv i ty was measured using a TRI-CARB Liquid
Scint i l la t ion Analyzer (Model 1900CA) wi th 5 ml of sc int i l la t ion l iquid (Ul t ima
GOLD, Packard) p lus 5 – 20 µ l sample. The mole rat io of [ 1 4C] bound per
tubul in d imer was calculated for each sample. For 1,5- IAEDANS modi f ied
tubul in samples, 8 or 10 µM modif ied tubul in solut ions were made ( f rom the
pooled f ract ion) in 0.1 M Tr isHCl, pH 8.0 buf fer and the absorbance spectra
(Cary 300 spectrophotometer) recorded for each sample. AEDANS2 has an
absorbance maximum at 330 nm at pH 8.0 wi th an ε = 5.7 x 103 M- 1 cm- 1 .
From the O.D. at 330 nm and the ext inct ion coeff ic ient the concentrat ion of
bound AEDANS to tubul in d imer was est imated. In addi t ion, t ime courses of
syn -monobromobimane (λe x=392 nm, λe m=480 nm, at a mole rat io of 67:1)
(16) and Thioglo 1 (λe x=379 nm, λe m=510 nm, at a mole rat io of 40:1) (17)
modi f icat ions of tubul in were done at room temperature by fo l lowing the
f luorescence of the product in a Perkin-Elmer LS-50B f luor imeter using 3mm
masked cel ls .
Distr ibut ion of label ing between α - and β - tubul in : The [1 4C]-modi f ied tubul in
samples were subjected to e lectrophoresis in 10% polyacrylamide gels (1.0 mm,
2.0 mm and 3.0 mm thick gels were used) to separate α - and β -subuni ts as
descr ibed by Knipl ing et a l . (18). Af ter the run, the gels were equi l ibrated in the
t ransfer buf fer (10 mM CAPS(3-[cyclohexylamino]-1-propanesul fonic acid) in 20%
methanol , pH 11) for 1-2 hr , and t ransferred to PDVF membrane ( Immobi lon PVDF
from Mi l l ipore) by apply ing 1-1.5 mA/cm2 over 5-8 h using Pharmacia LKB-
Mult iphor I I uni t . The membranes were ai r dr ied overnight and exposed to BAS-MS
Phosphor Imaging Plates for 10-20 days at room temperature. Then the Imaging
Plates were scanned in an FLA3000G Image Analyzer (Fuj i f i lm T M I&I Imaging, Fuj i
Medical Systems, USA, Inc). The IAEDANS modif ied tubul in samples were
subjected to electrophoresis in 10% polyacrylamide gels (3.0 mm thick) . The
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f luorescent gel bands were imaged using FluorChem T M8000-Advanced
Fluorescence, Chemi- luminescence, and Vis ib le Imaging Software. The gel was
exci ted at 302 nm to see the emission at 490 nm.
Trypsin Digest ion - The modi f ied tubul in samples (0.5–1.0 mg) were digested
wi th t rypsin-TPCK2 (1:20 weight rat io) at 37°C for 15-24 h in 0.1 M Tr is buf fer pH
8.5 wi th 5 mM CaCl2 . The t rypsin digests of modi f ied tubul in were subjected to
electrophoresis in 16% or 10-20% Tr is-Tr ic ine Novex precast gels.
Pept ide separat ion using C18 RP-HPLC2 - In order to obtain a higher y ie ld of
labeled pept ides we used a preparat ive (250 mm X 10 mm, pore s ize 300 Å,
part ic le s ize 10 µ) C18-RP column. 500 – 700 µg of the protein digest ( in 150-
200 µ l ) contain ing 4-6 M guanidine HCl, was sonicated and centr i fuged before
in ject ion into the column. A Perkin-Elmer Ser ies 410 LC Pump with a LC-95
UV/Vis ib le spectrophotometer was used to apply solvent gradients. We used
methanol instead of acetoni t r i le and the f ract ionat ion of labeled tubul in d igest
was achieved by apply ing the fo l lowing gradient ( f low rate 1.0 ml/min): Step 0 –
[ (5% methanol + 95% water) , 5 mM ammonium acetate] for 20 min; Step 1 – [ (5%
methanol + 95% water) , 5 mM ammonium acetate] to [ (50% methanol + 50%
water) , 5 mM ammonium acetate] over a per iod of 150 min; Step 2 – [ (50%
methanol + 50% water) , 5 mM ammonium acetate] to [ (95% methanol + 5%
water) , 5 mM ammonium acetate] over a per iod of 45 min; Step 3 – [ (95%
methanol + 5% water) , 5 mM ammonium acetate] for 40 min. This gradient gave
us good reproducibi l i ty and recovery of radioact iv i ty (greater than 80%). A C12-
RP column (50 x 4.6 mm, pore s ize 90 Å, part ic le s ize 4 µ) was used for fur ther
pur i f icat ion of pept ides. The absorbance at 214 nm was moni tored; the peaks
were col lected manual ly and counted.
Mass Spectra and Sequencing - The radioact ive and the f luorescent peaks were
concentrated on a Speedvac instrument and sent for MALDI-TOF (Matr ix-Assisted
Laser Desorpt ion/ Ionizat ion – Time Of F l ight) and N-terminal sequence analysis to
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the Macromolecular Structure Faci l i ty , Michigan State Universi ty. The masses of
the pept ides were calculated f rom the Protein and Pept ide Software developed by
Dr. Lewis Pannel l , NIH (ht tp: / /sx102a.niddk.nih.gov/pept ide).
Structure generat ion using RASMOL : RASMOL (19) was used to v isual ize and
generate tubul in structure (PDB Code: 1JFF).
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RESULTS
Kinet ics. I t has been proposed that several SH groups of tubul in may form
disul f ide bonds (20), but in the hands of most invest igators the 20 SH groups (12 in
α and 8 in β tubul in) are readi ly shown to be reduced, and the rat brain tubul in used
here consistent ly y ie lds >19 SH groups by t i t rat ion wi th excess DTNB in the absence
of any denatur ing agent. Moreover, the electron di f f ract ion structure shows no
disul f ide bonds (21). The t ime courses of th ioether format ion wi th iodoacetamide,
IAEDANS or the maleimides were compared in an excess of reagent ( reagent to
tubul in = 50:1, or 2.5:1 per SH group, at pH 6.9 and 37°C (Fig. 1A). Both SN2
displacing reagents, iodoacetamide and IAEDANS2, showed a s low progressive
increase in the number of cysteines react ing over a per iod of 3 h. Of considerable
interest is the f inding that the bulky IAEDANS2 reacted at the same rate as
iodoacetamide. Another reagent in th is c lass is syn -monobromobimane (16), whose
progress curve can by fo l lowed by f luorescence (Ex = 392 nm, Em = 480 nm,
quantum yie ld 0.2-0.3) as the reagent i tsel f is negl ig ib ly f luorescent. The in i t ia l rate
of react ion is not much faster than iodoacetamide and the extent of react ion is
comparable. As has been reported for smal l th io ls (16) the react ion is pH sensi t ive
and is a l inear funct ion of the [OH-] concentrat ion (over the pH range avai lable due
to the pI of the protein) (data not shown). As might be expected, monochlorobimane
reacts much more s lowly than i ts bromo congener (data not shown). Unfortunately,
monobromobimane is subject to photolysis leading to f luoresecent products, hence
minimal l ight exposure and careful b lank correct ions are cr i t ical at a l l t ime points.
Since separat ion of excess reagent is requi red, we have not fur ther pursued th is
label ing procedure. Nevertheless, a l l th ree reagents react wi th ~9 of the 20 SH
residues in 3 h (Fig 1A).
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Thioether format ion wi th maleimides occurs by nucleophi l ic addi t ion to a
double bond rather than by nucleophi l ic d isplacement. As shown in Fig. 1A ( top
curves), th is is a much faster and more extensive react ion. Thus, at 15 min and
37°C, v i r tual ly a l l of the cysteines have reached the plateau value seen at 2 h.
3-4 SH groups did not react over the 2 h t ime per iod. Interact ion wi th a f luorescent
maleimide analogue, Thioglo 1 (17), occurs at an equal ly rapid rate under these
condi t ions and again ~ 3-4 cysteines fa i led to react in the t ime al lowed. I t is of
interest that here, too, a bulky f luorophore in no way impeded access to 16-17
cysteines of tubul in; nor does the f luorophore s igni f icant ly accelerate interact ion as
has been noted for long-chain alkylmaleimides on proteins but not smal l th io ls (22).
Thus, whi le react ion condi t ions are not ident ical , i t is apparent that th ioether
format ion by nucleophi l ic d isplacement is substant ia l ly s lower than by addi t ion to
double bonds. This d i f ference in rates has been observed previously for smal l
th io ls; second order rate constants d i f fer by between 1 and 2 orders of magni tude
(23-25).
There are c lear ly fast- and s low-react ing SH groups in tubul in; the lat ter become
fast upon denaturat ion wi th urea such that the fu l l increase in f luorescence occurs
v i r tual ly instantaneously (data not shown) . With progressively increasing urea
concentrat ions two react ion c lasses ( in i t ia l s lopes) are observed – a re lat ively s low
rate up to ~1.5 M urea and a faster rate wi th urea >1.5 M.
Since the main object ive of the present study is to d iscover the locat ion of the
most react ive cysteines of tubul in, i t is necessary to devise condi t ions for at ta in ing
l imi ted stoichiometr ies by using low mole rat ios of reagent to tubul in, low
temperatures or low pH. To minimize reac t ions wi th less react ive cysteines we used
mole rat ios of 1:2 at 37°C at pH 6.9 for vary ing t imes. Under these condi t ions the
avai lable iodoacetamide was not used up at 3 h (Fig. 1B) and whi le there was very
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low incorporat ion at ear ly t ime intervals (e.g. 0.09 SH/dimer) , these amounts proved
to be useful for fur ther analysis as discussed below.
I t is wel l known that tubul in decays at 37°C (26, 27). Therefore, to minimize
any contr ibut ion f rom denaturat ion to the accessibi l i ty or react iv i ty of tubul in
cysteines, exper iments were carr ied out at 4°C wi th 30 µM tubul in at mole rat ios of
reagent/ tubul in of 1:5 to 50:1 at pH 6.9 for prolonged per iods. As shown in Fig. 1C
the 8 h incorporat ion rose gradual ly as a funct ion of the mole rat io for both
iodoacetamide and IAEDANS, but never exceeded 4 SH/dimer. These samples also
served as samples for t rypt ic pept ide analys is. About twice as much subst i tut ion
occurred wi th NEM.
Distr ibut ion of Label between α - and β- tubul in. In prel iminary exper iments to
compare the distr ibut ion of label between the two tubul in monomers, we used 0.06-
0.6 moles of [1 4C]-NEM per d imer, 0.05-0.3 moles[1 4C]- iodoacetamide per d imer, or
0.5-4.0 moles IAEDANS per dimer. 10% sodium dodecyl sul fate polyacry lamide gels
were used to separate α - and β - tubul in fo l lowed by t ransfer to PVDF membranes,
phosphor imaging (Figs. 2A & B), or f luorescence analysis (Fig. 2C). Under var ious
incubat ion condi t ions using di f ferent temperatures, t imes or mole rat ios, no
condi t ions could be found that led to unique label ing of only one monomer at the
expense of the other. Even wi th mole rat ios as low as 0.06 (Fig. 2A) of label per
d imer both monomers were labeled. Ident ical resul ts were obtained wi th [ 1 4C]-NEM
(Fig. 2B) or wi th IAEDANS (Fig. 2C). Al though the l i terature deals almost exclusively
wi th modi f icat ion of SH groups on β - tubul in, the α - tubul in SH groups appear to react
at least as v igorously and, in the case of IAEDANS, α - tubul in label ing exceeds that
of β - tubul in. This suggests possibly fast , but part ia l , react ions on several cysteine
residues whose react iv i ty cannot be dist inguished by these reagents; moreover, i t is
c lear that incorporat ion of one equivalent of any of these three sul fhydryl reagents
cannot be unambiguously interpreted in terms of a s ingle cysteine residue.
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Low resolut ion mapping of the most react ive tubul in cysteines- The
modi f ied tubul in, d igested wi th t rypsin-TPCK for 24 h at 37°C, was separated on a
16% Tr is-Tr ic ine gel . 12 Cysteines out of 20 are present in larger (≥ 2.4 kD) t rypt ic
pept ides that could be ident i f ied in 16% Tr is-Tr ic ine gels. The fo l lowing four α -
tubul in t rypt ic pept ides contain 7 cysteines: (1) residues 3-40 (3.8 kDa) → C4, C20
& C25, (2) residues 125-156 (3.2 kDa) → C129, (3) residues 167-214 (4.8 kDa) →
C200 & C213, and (4) residues 281-304 (2.4 kDa) → C295. The fo l lowing three β -
tubul in t rypt ic pept ides contain 5 cysteines, (1) residues 123-154 (3.2 kDa) →
C129 & C131, (2) residues 175-213 (3.9 kDa) → C201 & C211, and (3) residues
217-241 (2.5 kDa) → C241. Most of the bound radioact iv i ty (75 to 80%) was lost
dur ing the electrophoresis. This c lear ly indicates that the smal ler t rypt ic pept ides
contained the most react ive cysteines. So the fo l lowing 11 tubul in cysteines could
be el iminated safely as the react ive cysteines: C4:α , C20:α , C25:α ,C129:α ,
C200:α , C213:α , C295:α , C129:β , C131:β , C203:β , and C213:β . The fo l lowing 9
cysteines C315:α , C316:α , C305:α , C347:α , C376:α , C12:β , C305:β , and C356:β1
p lus C241:β1 should contain the fast-react ing cysteines of tubul in.
Local izat ion of the Most React ive Cysteine Residues- Trypt ic pept ides
f rom tubul in, labeled at low mole rat ios, were analyzed by HPLC, mass
spectroscopy and N-terminal sequencing. To ident i fy the most react ive tubul in
cysteines wi th iodoacetamide, the fo l lowing two samples were used: tubul in (30
µM) was incubated (1) wi th [ 1 4C]- iodoacetamide (15 µM, 56 dpm/pmol) and (2)
wi th [1 4C]- iodoacetamide (1.5 mM, 2.44 dpm/pmol) for 60 min at 37°C. The
samples were processed according to the procedure descr ibed in the methods
sect ion. The iodoacetamide to tubul in ra t io was 1:2 in the former case and 50:1
in the later , the moles [1 4C] incorporated were 0.16 and 5.6, respect ively. In
order to obtain a higher y ie ld of labeled pept ides, (1) we digested the whole
tubul in rather than separat ing and pur i fy ing α - and β - tubul ins, and (2) used a
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preparat ive (250 mm X 10 mm) C18 column. The format ion of soluble pept ide
aggregates impeded the progress of the exper iment, so a high concentrat ion of
about 4-6 M guanidine HCl was used for sample preparat ions. The common
acetoni t r i le gradient d id not g ive reproducible resul ts wi th good recovery of
radioact iv i ty . We used methanol instead of acetoni t r i le for the f ract ionat ion of
labeled tubul in d igest . This gradient y ie lded good reproducibi l i ty and recovery of
radioact iv i ty
(> 80%).
F igure 3A shows the HPLC-chromatogram of a digest of tubul in
modi f ied wi th [1 4C]- iodoacetamide to a mole rat io of 0.16 per d imer. We ident i f ied
f ive peaks, labeled as 1*, 2*, 3*, 4* and 5*, wi th s igni f icant radioact iv i ty (Fig.
3A). The inset in Fig. 3A shows the radioact iv i ty of the peaks. Even though the
mole rat io of [ 1 4C] bound per tubul in d imer was 0.16, we observed f ive
radioact ive peaks, later ident i f ied as three coming f rom α - tubul in and two f rom β -
tubul in (see below). Fig. 3B shows the HPLC-chromatogram of [ 1 4C]-
iodoacetamide-reacted tubul in wi th a mole rat io of 5.6 per d imer. Again the
radioact iv i ty was local ized on the same f ive peaks as at low mole rat ios. Thus,
there are at least f ive fast-react ing cysteines present in tubul in and al l of them
react wi th iodoacetamide even at substoichiometr ic label ing.
Simi lar resul ts were obtained when tubul in was labeled wi th [ 1 4C]-NEM.
Fig. 4A shows chromatograms of tubul in modi f ied at a mole rat io of [ 1 4C] to d imer
of 0.45. Under these condi t ions 7 labeled peaks could be ident i f ied (*) despi te a
total label ing stoichiometry of <<1 NEM/dimer. This greater number of modi f ied
cysteines is expected because of the greater react iv i ty of NEM. When a much
higher mole rat io of 5.1 NEM/dimer was used, only one addi t ional labeled pept ide
was obtained as shown in Fig. 4B. In addi t ion to the peaks labeled wi th
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iodoacetamide above, several of these peaks could not be ident i f ied on the basis
of their masses and these were not pursued fur ther.
Our fa i lure to ident i fy a s ingle ‘most react ive’ cysteine using ei ther
iodoacetamide or N–ethylmaleimide suggested the use of a less potent reagent
such as chloroacetamide. When [ 1 4C]-chloroacetamide (1.5 mM with 30 µM
tubul in for 3 h at 37°C) was used for k inet ic analysis, the moles of [ 1 4C]
incorporated per d imer were only 0.2 as compared to 8 when iodoacetamide was
used under the same condi t ions. When 1/10 as much chloroacetamide was used
0.02 moles of [1 4C] were incorporated per d imer. Analysis of the t rypt ic pept ides
revealed a s ingle radioact ive peak corresponding to C347:α ( that was also
labeled by the other reagents as shown above) (Fig. 5 inset) . I t is c lear that by
th is approach the one most react ive cysteine could be selected f rom the other
fast-react ing cysteine residues.
Masses and sequences of pept ides bear ing the most react ive cysteine
residues. The t rypt ic pept ides obtained f rom IAEDANS2-, iodoacetamide-, and
NEM2-modi f ied tubul in af ter HPLC2 separat ion (Table 1–column 3) are compared
wi th their calculated masses (Table 1–column 2). These values are in good
agreement. Subsequent N-terminal sequencing revealed that each pept ide had
one or two unident i f ied residues in the cysteine posi t ion of the pr imary sequence.
This accounted for the expected mass of the part icular modi f icat ion of the
pept ide. Table 1–A l is ts the four IAEDANS-modif ied pept ides contain ing f ive
cysteines: C305:α , C315-C316:α , C347:α and C354:β . For reasons we don’ t
understand at present, iodoacetamide modi f ied f ive pept ides (see also Fig. 3A
and 3B) but one (peak 1) could not be ident i f ied. The modi f ied cysteines are:
C305:α (peak 2) , C347:α (peak 4) , C241:β (peak 5) , and C356:β (peak 3). Seven
pept ides were labeled by NEM2 (Fig. 4A and 4B) as shown in Table 1–C:
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C305:α (peak 2) , C315-316:α (peak 3) , C347:α (peaks 6 & 7), C376:α (peak 5),
C241:β (peak 8)1 , and C356:β (peak 4)1 . Again we could not ident i fy peak 1. Note
that dur ing t rypsin digest ion at pH 8.5, the NEM group may undergo hydrolysis
wi th a mass increase of 18 (H2O) leading to two entr ies in column 2, Table 1C.
In sum these data show that the most react ive β - tubul in cysteines are C241(239)
and C356(354) as has been found by others (see Discussion). α -Tubul in has f ive
react ive cysteines: C305, C315, C316, C347 and C376.
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DISCUSSION
A major factor determining the react iv i ty of cysteines is their d issociat ion to
the th io late anion. Increased th io l d issociat ion can be promoted by one or more
interact ions in which the excess electron densi ty of the th io late product is stabi l ized
by interact ions wi th posi t ive charge. Any fu l l or part ia l posi t ive charges in the
neighborhood (<6.5 Å) wi l l tend to stabi l ize the th io late anion, thus lower i ts pKa and
increase i ts react iv i ty . Short- range ef fects resul t ing f rom hydrogen bonds and f rom
main chain carbonyl carbon atoms may wel l contr ibute to th io l react iv i ty but cannot
be useful ly analyzed f rom the 3.5Å structure current ly avai lable. They have not
been considered in the present study. The other posi t ive charges can der ive f rom
the fo l lowing:
1. Coulombic stabi l izat ion of the th io late anions by posi t ively charged amino
acid s ide chains of His, Lys and Arg at d istances not to exceed 6.5Å. Snyder et a l .
(28) have shown wi th cyanogen bromide-generated pept ides of smal l proteins that
react ion rates of cysteine wi th DTNB vary over many orders of magni tude as a
funct ion of the charge of the nearest neighbor amino acid s ide chains in the pr imary
sequence. These ef fects are markedly reduced at h igh ionic strength, at test ing to
the electrostat ic nature of the act ivat ion of the th io ls by the local environment. The
die lectr ic environment thus has a considerable ef fect on the extent of these
interact ions. Several studies wi th smal l pept ides have conf i rmed th is ef fect wi th S-
palmitoylat ion (29, 30) . However, the distance between the posi t ive charge and the
th io late anion is indeterminate in these pept ides.
2. Cysteine-aromat ic interact ions (31). The π e lectron c loud on the faces of the
aromat ic r ing interact strongly wi th cat ions. With the large number of aromat ic
residues in tubul in, i t is not surpr is ing that there are 6 readi ly ident i f ied cat ion-π
interact ions. These are: F343:α /K336:α , Y399:α /R402:α , F395:α /R422:α ,
F92:β /R79:β , F399:β /K402:β and W346:β /K401:α . The obverse of th is e lectron
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distr ibut ion impl ies that r ing edges are re lat ively posi t ively charged and capable of
interact ing wi th anions, a l though these interact ions may not be as strong as the
cat ion-π interact ion. I t has been shown (32, 33) that many cysteines of proteins are
c lose enough to aromat ic r ings to interact in th is way. In model hel ical pept ides
interact ion between phenylalanine and cysteine in the proper or ientat ion ( i , i+4)
contr ibutes s igni f icant ly to hel ix stabi l i ty (34) . Al though the die lectr ic constants of
protein inter iors are di f f icul t to ascerta in, the electr ical potent ia l is a reciprocal
funct ion of the die lectr ic constant , hence the electrostat ic ef fects may be enhanced
by low local d ie lectr ic environments; the nearby aromat ic residues may contr ibute to
th is . There is, however, some disagreement regarding the detai ls of th is interact ion
(35).
3. The N-terminus of an α -hel ix d ipole is posi t ively charged and may thus
stabi l ize a nearby th io late (36). From model α hel ices Kortemme and Creighton (37)
have measured a decrease in th io l pKa of 1.6 pH uni ts. In selected cases two or
even three hel ices may stabi l ize the same thio late (36).
As ment ioned above, i t was not possib le wi th these three reagents under
these condi t ions to ident i fy a unique cysteine residue. Five pept ides that
contained 6 of the 20 cysteines were the most easi ly labeled. They were C315 +
C316, C347, and C376 from α - tubul in, and C241 and C356 f rom β - tubul in (Fig.
6) . The numbering fo l lows that of the electron di f f ract ion structure (21) and is
equivalent to C239:β and C354:β in the pr imary sequence.
Cysteine residue C376:α , a l though near ly bur ied, appears to be act ivated by
R320:α and Y272:α (127°) (Fig. 6A). The sul fur to r ing-edge angle is l is ted only in
the legend but not the Figs. ( in parentheses). Whether or not pr ior subst i tut ion of
another SH group promotes exposure of C376 remains to be determined. Note that
i t was th is residue in p latelet tubul in that was the substrate for palmitoylat ion (10)
but is not the most react ive toward reagents forming th ioethers in rat brain tubul in.
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I t is of interest that His residues are only rarely involved; C20:α has a His residue at
4.9 Å; hel ix d ipoles appear not to part ic ipate in the act ivat ion of th is group of
cysteines.
The most extensively labeled th io l belonged to C347:α , which contained over
hal f of the incorporated label (Fig. 6B). Factors that may have contr ibuted to i ts
react iv i ty are probable exposure to the solvent in the f ree dimer, the presence of
K336 at 6.1Å, and the presence of two posi t ively charged aromat ic r ing edges f rom
F343 and W346 both at favorable angles, 146° and 127°, respect ively. I t is not
known whether th is re lat ionship, present in the polymer, remains the same in the
f ree dimer. Moreover, C347:α is the most exposed of the react ive cysteines.
The α pept ide contain ing two SH groups, C315:α and C316:α , was also
labeled by the sul fhydryl reagents (Fig. 6C). The two SH groups are separated by 9
Å (data not shown), too far for ready disul f ide format ion. A remarkable feature of
the C315:α SH environment is a c luster of four aromat ic residues. Three of these
are posi t ioned at favorable angles for r ing edge contact – F343(146°) , F296(156°) ,
and Y312(132°). F351 is unfavorably posi t ioned at near ly a r ight angle (80°) .
Al though we could not separately analyze C315 and C316, one would predict that
C316 would be the less react ive SH of the two because K352:α is at 6.3Å and F155
subtends an angle of 92°.
Two β - tubul in SH groups react ing at low reagent/ tubul in rat ios were C241:β
and C356:β . These groups can be cross- l inked by bi funct ional reagents (11) and,
whi le far apart in the pr imary sequence, both are act ivated by the same argin ine
(R320:β ) , hence we represented them together in Fig. 6D. In addi t ion, these
residues are act ivated by F244:β whose r ing edge is posi t ioned at favorable react ion
angles: 160° to C241 and 141° to C356. Al though C241:β is nearer to a formal
posi t ive charge than any other SH group of tubu l in, i t is not the most react ive. This
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may possibly be due to the fact that access to reagent is constra ined by the
presence of C241 in hel ix 7 and R320 in a β sheet rather than a loop. Note that
these residues are also c lose to the colchic ine-binding s i te, which may explain the
inhib i tory ef fect of s i te occupancy on cross- l ink ing by bi funct ional reagents (11). In
th is respect i t is interest ing that certa in natural product ant imicrotubule agents,
quinones (38-40) and benzophenanthr id ines (41) inhib i t microtubule assembly
through interact ion wi th SH groups.
The inact iv i ty of a number of the cysteine th io l groups is a lso amenable to
e lectrostat ic analysis. Al l s ix remaining cysteines of β - tubul in f ind themselves in the
v ic in i ty of one or more carboxylate groups that are expected to suppress
dissociat ion of the th io ls (Fig. 7) . C129:β and C131:β , which are surface accessib le,
nevertheless do not react wi th the SH reagents under our condi t ions – C129:β is de-
act ivated by E3:β whereas C131:β is surrounded by three carboxylates which
appears to be enough to overcome any act ivat ing ef fects of R164:β (Fig. 7A). Fig.
7B shows that C203:β is negat ively control led by D205:β despi te two phenylalanines
in the environment. A s imi lar charge antagonism obtains in Fig. 7C where C12:β ,
a l though at the N-terminal end of hel ix 1, is inhib i ted by a nearby phosphate as wel l
as being blocked by GDP (Fig. 7C). Again in Fig. 7D, asparty l charges f rom D226
appear to overcome any ef fects f rom Y210. By contrast to β - tubul in we are able to
explain de-act ivat ion of only one α - tubul in cysteine – namely the surface exposed
C129:α (Fig. 7E).
Another cr i t ical factor in the react ion of cysteines is the ‘ react iv i ty ’ of the
modi fy ing reagents. In a number of studies negat ive charge on the reagent has
compl icated interpretat ion (13, 28-30). In the present study we have used two
uncharged alkylat ing reagents and IAEDANS2 wi th a sul fonate group, and we have a
measure of the distance between charge and SH permit t ing a bet ter est imate of
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some act ivat ing factors. The fact the IAEDANS2 is at least as act ive as
iodoacetamide suggests that e i ther i ts negat ive charge is far away f rom the
interact ing surface or that the ani l ino ni t rogen mit igates the negat ive charge ef fect .
I t a lso suggests that bulky groups, as in IAEDANS, monobromobimane or Thioglo 1,
have ready access to non-surface cysteines. Under these condi t ions the common
reagents used were too act ive to permit d ist inct ions between the most react ive
cysteines. Thus, a unique most act ive th io la te could be ident i f ied only by use of the
poor ly act ive chloroacetamide. This residue was C347:α , the most react ive of a l l
tubul in th io ls studied. Severa l other reagents have ident i f ied C241:β1 as the only
react ive th io l – these are, however, s i te-di rected by binding in the area of the
colchic ine binding s i te (4, 5, 42).
Considerable di f f icul ty exists in the in terpretat ion of the solvent accessibi l i ty
of the cysteine residues of tubul in. Us ing DTNB, many invest igators have shown
that a l l 20 Cys residues react . Of these, ~5 reacted very rapidly whereas the
remaining 15 were more s lowly react ing. Roychowdhury et a l . (43) have interpreted
th is in terms of 5 surface-exposed cysteines and 15 residues ‘bur ied’ , as calculated
according to Fraszkiewicz and Braun (44). The high act iv i ty of DTNB precludes
rate di f ferent iat ion wi th in these groups. They also showed only minor c i rcular
d ichroism changes f rom DTNB modi f icat ion, reportedly excluding opening up of
bur ied residues by pr ior modi f icat ion of fast-react ing residues (43). The resul ts
obtained in the present study as wel l as var ious in previous reports, most ly on β -
tubul in, are not consistent wi th that interpretat ion. Four of the f ive fast-react ing
cysteines are ‘bur ied’ according to th is calculat ion, and only one is solvent-exposed.
I t is noteworthy in th is respect that a 95% bur ied cysteine residue in barstar has
fu l ly reacted wi th DTNB in 10 min (45). Moreover, several surface-exposed
cysteines, e.g. C129:β and C131:β , are not modi f ied by l imi t ing iodoacetamide. As
shown above, their e lectrostat ic environment is inhib i tory due to the presence of
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carboxylates. Thus, e lectrostat ics appear to outweigh exposure. The fa i lure of
solvent exposure to explain react iv i ty might be due to one of the fo l lowing:
1. The dimer structure der ived f rom electron di f f ract ion of z inc sheet polymers
(21) may not accurately descr ibe the f ree dimer in solut ion. Addi t ional
evidence der ives f rom the f inding (Fig. 1) that the rate of subst i tut ion by
iodoacetamide is the same as for IAEDANS and a bulky NEM der ivat ive reacts
as rapidly as NEM.
2. Calculat ions of solvent exposure of tubul in f rom the 3.5 Å structure are not
re l iable.
3. In i t ia l th io l modi f icat ions may ‘open up’ domains as has been found for
lys ine:N6-hydroxylase (46) but which may not be suf f ic ient for detect ion, e.g.
by c i rcular d ichroism.
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REFERENCES 1. Shivanna, B. D., Mejillano, M. R., Williams, T. D. and
Himes, R. H. (1993) J. Biol. Chem. 268, 127-132
2. Gupta, M. L., Bode, C. J., Dougherty, C. A., Marquez, R.
T. and Himes, R. H. (2001) Cell Motil. Cytoskel. 49, 67-
77
3. Uppuluri, S., Knipling, L., Sackett, D. L. and Wolff, J.
(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11598-11602
4. Bai, R., Lin, C. M., Nguyen, N. Y., Liu, T.-Y. and
Hamel, E. (1996) Biochemistry 28, 5606-5612
5. Shan, B., Medina, J. C., Santha, E., Frankmoelle, W. P.,
Chou, T.-C., Learned, R. M., et al. (1999) Proc. Natl.
Acad. Sci. U. S. A. 96, 5686-5691
6. Zambito, A. M. and Wolff, J. (1997) Biochem. Biophys.
Res. Commun. 239, 650-654
7. Caron, J. M. (1992) Mol. Biol. Cell 8, 621-636
8. Wolff, J., Zambito, A. M., Britto, P. J. and Knipling,
L. (2000) Prot. Sci. 9, 1357-1364
9. Zambito, A. M. and Wolff, J. (2001) Biochem. Biophys.
Res. Commun. 283, 42-47
10. Ozols, J., and Caron, J. M. (1997) Mol. Biol. Cell 8,
637-645
11. Luduena, R. F. and Roach, M. C. (1991) Pharmacol.
Ther. 49, 133-152
12. Friedman, M. (1973) in The Chemistry and Biochemistry
of the Sulfhydryl Group in Amino Acids, Peptides and
Proteins Pergamon Press, Oxford
13. Brocklehurst, K. and Little, G. (1973) Biochem. J.
133, 67-80
14. Wolff, J., Knipling, L. and Sackett, D. L. (1996)
Biochemistry 35, 5910-5920
by guest on April 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
24
15. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia,
A. K., Gartner, F. H., Provenzano, M. D., et al. (1985)
Anal. Biochem. 150, 76-85
16. Radkowski, A. E. and Kosower, E. M. (1986) J. Am.
Chem. Soc. 108, 4527-4531
17. Wright, S. K. and Viola, R. E. (1998) Anal. Biochem.
265, 8-14
18. Knipling, L., Hwang, J. and Wolff, J. (1999) Cell
Motil. Cytoskel. 43, 63-71
19. Sayle, R. and Milner-White, E. J. (1995) Trends in
Biochem. Sci. (TIBS) 20, 374-376
20. Chaudhuri, A. R., Khan, I. A. and Luduena, R. F.
(2001) Biochemistry 40, 8834-8845
21. Löwe, J., Li, H., Downing, K. H. and Nogales, E.
(2001) J. Mol. Biol. 313, 1045-1057
22. Denicola-Seoane, A. and Anderson, B. M. (1990)
Biochim. Biophys. Acta 1040, 84-88
23. Gorin, G., Martic, P. A. and Doughty, G. (1966) Arch.
Biochem. Biophys. 115, 593-597
24. Hanson, H. and Hermann, P. (1958) Bull. Soc. Chim.
Biol. 40, 1835-1847
25. Steenkamp, D. J. (1993) Biochem. J. 292, 295-301
26. Wilson, L. (1970) Biochemistry 9, 4999-5007
27. Prakash, V. and Timasheff, S. N. (1992) Arch. Biochem.
Biophys. 295, 137-154
28. Snyder, G. H., Cennerazzo, M. J., Karolis, A. J. and
Field, D. (1981) Biochemistry 20, 6509-6519
29. Bélanger, C., Ansanay, H., Quanbar, R. and Bouvier, M.
(2001) FEBS Lett. 499, 59-64
30. Bizzozero, O. A., Bixler, H. A. and Pastuszyn, A.
(2001) Biochem. Biophys. Acta 1545, 278-288
by guest on April 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
25
31. Ma, J. C. and Dougherty, D. A. (1997) Chem. Rev. 97,
1303-1324
32. Morgan, R. S., Tatsch, C. E., Gushard, R. H., McAdon,
J. M. and Warme, P. K. (1978) Int. J. Peptide Prot. Res.
11, 209-217
33. Reid, K. S. C., Lindley, P. F. and Thornton, J. M.
(1985) FEBS Lett. 190, 209-231
34. Viguera, A. R. and Serrano, L. (1995) Biochemistry 34,
8771-8779
35. Pal, D. and Chakrabarti, P. (1998) J. Biomol. Struct.
Dynam. 15, 1059-1071
36. Hol, W. G. J. (1985) Prog. Biophys. Mol. Biol. 45,
149-195
37. Kortemme, T., and Creighton, T. E. (1995) J. Mol.
Biol. 253, 799-812
38. Müller, W. E. G., Dogovic, N., Zahn, R. K., Maidhof,
A., Diehl-Seifert, B., Becker, C., et al. (1985) Basic
Appl. Histochem. 29, 321-330
39. O’Brien, E. T., Asai, D. J., Groweiss, A., Lipshutz,
B. H., Fenical, W., Jacobs, R. and Wilson, L. (1986) J.
Med. Chem. 29, 1851-1855
40. Pfeiffer, E. and Metzler, M. (1996) Chem. Biol.
Interactions 102, 37-53
41. Wolff, J. and Knipling, L. (1993) Biochemistry 32,
13334-13339
42. Legault, J., Gaulin, J-F., Monneton, E., Boldue, S.,
Lacroix, J., Poyet, P. and C.-Gaudreault, R. (2000)
Cancer Res. 60, 985-992
43. Roychowdhury, M., Sarkar, N., Manna, T.,
Bhattacharyya, S., Sarkar, T., BasuSarkar, P., Roy, S.
and Bhattacharyya. B. (2000) Eur. J. Biochem. 267, 3469-
3476
by guest on April 5, 2018
http://ww
w.jbc.org/
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nloaded from
26
44. Fraszkiewicz, R. and Braun, W. (1998) J. Comp. Chem.
19, 319-333
45. Ramachandran, S., Rami, B. R. and Udgaonkar, J.B.
(2000) J. Mol. Biol. 297, 733-745.
46. Dick, S., Siemann, S., Frey, H. E., Lepock, J. R. and
Viswanatha, T. (2002) Biochim. Biophys. Acta 1594, 219-
233.
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FIGURE LEGENDS
Figure 1: The react ion of sul fhydryl reagents wi th tubul in. (A) High mole rat io of
reagent to tubul in: Tubul in (30 µM in 334 µ l ) samples were incubated wi th the
sul fhydryl reagents ( ) syn -monobromobimane, 67:1, ( ) [ 1 4C]-NEM, 50:1, ( )
[ 1 4C]- iodoacetamide, 50:1, ( ) 1,5- IAEDANS, 50:1, ( ) Thioglo 1, 40:1, at 37°C in
the dark. (B) Low mole rat io of reagent to tubul in: Tubul in (30 µM in 334 µ l )
samples were incubated wi th ( ) [ 1 4C]-NEM, 1:2, and ( ) [ 1 4C]- iodoacetamide, 1:2
at 37°C in the dark. (C) Tubul in (30 µM in 334 µ l ) samples were incubated wi th
vary ing concentrat ions, 1:5, 1:2, 1:1, 3:1, 5:1 and 10:1, of ( ) [ 1 4C]-
iodoacetamide, ( ) 1,5- IAEDANS, and ( ) [ 1 4C]-NEM at 4°C in the dark for 8 h. At
regular t ime intervals, the react ion was stopped by adding β -mercaptoethanol and
processed as in Mater ia ls and Methods.
F igure 2: Distr ibut ion of labels between α - and β - tubul in subuni ts. Note that α -
tubul in is the upper and β - tubul in is the lower band. (A) Tubul in (30 µM) samples
were incubated wi th [ 1 4C]- iodoacetamide (15 µM, 56 DPM/pmol) at 37°C. The mole
rat ios were 0.06 ( lane 1, 15 min), 0.09 ( lane 2, 30 min), 0.16 ( lane 3, 60 min), 0.2
( lane 4, 90 min), 0.24 ( lane 5, 120 min), 0.28 ( lane 6, 150 min) and 0.33 ( lane 7,
180 min). 8 µg protein was loaded on each lane. (B) Tubul in (30 µM) samples
were incubated wi th [ 1 4C]-NEM (15 µM, 133 DPM/pmol) at 37°C and processed as
above. 30 µg protein loaded on each lane. The mole rat ios were 0.45 ( lane 1, 15
min) and 0.45 ( lane 2, 30 min). Also tubul in (30 µM) samples were incubated wi th
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varying [1 4C]-NEM concentrat ions at 4°C for 5 h. The mole rat ios were 0.06 ( lane
3, 1:10), 0.12 ( lane 4, 1:5) , 0.35 ( lane 5, 1:2) , 0.65 ( lane 6, 3:1) and 2.4 ( lane 7,
5:1) . (C) Tubul in (30 µM) was incubated wi th 1,5- IAEDANS with varying
concentrat ions at 4°C for 8 h. 30 µg protein was loaded on each lane (gel : 3.0 mm
and 10 lane). The mole rat ios were 0.5 ( lane 2), 0.8 ( lane 3), 1.1 ( lane 4), 1.73
( lane 5), 2.4 ( lane 6) and 8.0 ( lane 7); lane 1 – rat brain tubul in (RBT). The gel
was exci ted at 302 nm to see the emission at 490 nm.
F igure 3: Local izat ion of most react ive cysteines towards iodoacetamide. Tubul in
(30 µM) was incubated, wi th [ 1 4C]- iodoacetamide (15 µM, 56 DPM/pmol) or wi th
1.5 mM, 2.44 DPM/pmol for 60 min at 37°C. Samples were processed according to
Mater ia ls and Methods. Absorbance at 214 nm was moni tored, intensi ty is depicted
in mi l l ivol ts (mV); peaks were col lected and their radioact iv i ty was measured. (A)
Chromatogram of [ 1 4C]- iodoacetamide modi f ied tubul in wi th a mole rat io of 0.16,
and (B) chromatogram of [ 1 4C]- iodoacetamide modi f ied tubul in at a mole rat io of
5.6.
F igure 4: Local izat ion of most react ive cysteines wi th [ 1 4C]-NEM. Tubul in (30 µM)
was incubated, (1) wi th [1 4C]-NEM (15 µM, 120 DPM/pmol) for 15 min at 37° C or
1.5 mM, 2.47 DPM/pmol for 60 min at 37°C. At the end of the incubat ion per iod the
samples were processed according to the procedure descr ibed in Methods.
Absorbance at 214 nm was moni tored, peaks were col lected and their radioact iv i ty
was measured. (A) Chromatogram of [ 1 4C]-NEM modif ied tubul in wi th a mole rat io
of 0.45, and (B) Chromatogram of [ 1 4C]-NEM modif ied tubul in wi th a mole rat io of
5.1.
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Figure 5: Local izat ion of most react ive cysteines wi th [ 1 4C]-chloroacetamide.
Tubul in (30 µM) was incubated wi th [ 1 4C]-chloroacetamide (150 µM, 125
DPM/pmol) for 180 min at 37°C. Samples were processed as in Methods. The mole
rat io of bound [1 4C] to tubul in was 0.02. The modi f ied tubul in (1.0 mg) was
digested wi th t rypsin and separated on a C18 preparat ive HPLC column wi th a
water-methanol gradient . Absorbance at 214 nm was moni tored, peaks were
col lected and their radioact iv i ty was measured ( inset) .
F igure 6: Tubul in Cysteines in Posi t ive Surroundings. Structures were
generated using RASMOL. The tubul in d imer (accession number 1JFF) was
displayed as a r ibbon diagram ( in Grey) and speci f ic residues and/or speci f ic
regions were highl ighted in d i f ferent colors. The fo l lowing color scheme was
used: loops → magenta, β -sheets → cyan, and α -hel ix → yel low. Cys, Arg, Lys,
Asp, Glu, Phe, Tyr, Trp and His residues were displayed as wireframe models.
When a Cys residue belongs ( i ) to a α -hel ix , i t was displayed in yel low-
wireframe, ( i i ) to a β -sheet, i t was displayed in cyan-wireframe, and ( i i i ) to a
loop, i t was displayed in magenta-wireframe; so also wi th other residues. Sul fur
atoms, s ide chain ni t rogen atoms of Arg & Lys, and s ide chain oxygen atoms of
Asp & Glu were displayed as spacef i l l model in the CPK color scheme: S→dark
yel low (CPK), N→sky blue (CPK) and O→ red (CPK). Angles between sul fur and
the nearest r ing carbon are l is ted in parentheses. Hel ix (H), β sheet (B) and
loop (T) locat ions are in parentheses (21).
(A) Environment of C376:α ( in B10). C376:α has a posi t ive neighbor,
R320 (B8), and an aromat ic residue, Y272(127°, B7) at a d istance < 6.5Å.
(B) Environment of C347:α ( loop between H10 & B9). C347:α has a posi t ive
neighbor, K336 (H10), and two aromat ic residues, F343(146°) and W34(127°)
( loop between H10 & B9) at a d istance < 6.5Å. (C) Environment of C315:α -
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C316:α (B8). C316:α has a posi t ive neighbor, K352 (B9) and an aromat ic
residue, F255(92°) (H8). C315:α is surrounded by four aromat ic residues,
Y312(132°, B8), F296(156°, H9), F343(143°) ( loop between H10 & B9) and
F351(88°, B9). (D) Environment of C241:β and C356:β . Both C241(239):β
(H7) and C356(354):β (B9) share a posi t ive group, R320 (B8) and an aromat ic
residue, F241 (T7 loop). The angle between C241-sul fur to the plane of F244 is
160° and C356-sul fur to the plane of F244 is 141° .
F igure 7: Tubul in cysteines in negat ive surroundings. Color notat ions as in Fig. 6.
(A) Environments of C129:β and C131:β ( loop between H3 & B4). C129:β has a
negat ive neighbor E3 (N-terminus). C131:β has one posi t ive neighbor, R164 ( loop
between H4 & B5) and three negat ive neighbors, D130:β ( loop between H3 & B4),
D98:α (T3- loop) and E97:α (T3- loop). (B) Environments of C203:β (T6 loop) and
C305:β ( loop between H9 and H9’) . C203:β has one negat ive neighbor, D205 (T6-
loop) and two aromat ic residues, F267(131°, B7) and F388(143°, H11). C305:β
has two negat ive neighbors, D205 (T6- loop) and D306. (C) Environment of C12:β
(H1). C12:β has one negat ive neighbor, oxygen of GDP at 4.7Å. (D) Environment
of C213:β (H6). C213:β has a negat ive neighbor, D226 (H7) and an aromat ic
residue, Y210(171°, H6). (E) Environment of C129:α ( loop between H3 & B4).
C129:α has a negat ive neighbor, E3 (N-terminus).
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TABLE 1
MALDI-TOF and N-terminal sequencing data of tubul in cysteine pept ides modi f ied
by (A) 1,5- IAEDANS, (B) [1 4C]- iodoacetamide, and (C) [ 1 4C]-NEM. The weight
values were given for [M+H]+ ions: ( a ) 1 cysteine-modi f ied pept ide; ( b ) 2 cysteine-
modi f ied pept ide; ( c a n d d ) weight d i f ferences due to di f ferent isotypes; ( e ) for NEM,
the calculated weights were given for pept ides wi th unhydrolyzed and hydrolyzed
NEM moiety. The weight values, [M+H]+, of the unmodif ied pept ides were 490.6
(305-308:α ) , 1136.4 (312-320:α ) , 1528.8 & 1542.8 (340-352:α ) , 1809.1 (374-
390:α ) , 2653.1 (217-241:β ) , and 972.2 (351-359:β ) . The pept ide mixtures obtained
f rom the t rypsin-TPCK digest ion of modi f ied tubul in were f ract ionated by RP-HPLC
using a C18 preparat ive column. The peaks wi th s igni f icant radioact iv i ty f rom [1 4C]-
NEM- and [1 4C]- iodoacetamide-modi f ied tubul in, or wi th f luorescence f rom 1,5-
IAEDANS modif ied tubul in, were col lected, analyzed by MALDI-TOF and then by N-
terminal (5 or 6 residues) sequencing.
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FOOTNOTES
1Due to a di f ference in al ignment in the electron di f f ract ion structure of β -
tubul in, C241 corresponds to C239, and C356 corresponds to C354 in the l inear
sequence.
2The abbreviat ions used are: CPK, space f i l l ing atomic model according to
Corey, Paul ing and Kendrew; DPM, dis integrat ions per minute; DTNB, di th io-bis-
(2-ni t robenzoate) or El lman’s reagent; IAEDANS, 5-((( (2- iodoacety l )amino)ethyl) -
amino) naphthalene-1-sul fonic acid, and AEDAN is the f luorescent moiety that is
bound to protein and lacks the iodine; NEM, N-ethylmaleimide, RPHPLC, reverse
phase high performance l iquid chromatography; TPCK, L-1-tosylamide-2-phenethyl
chloromethyl ketone.
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TABLE 1 (A) Molecular mass of AEDANS = 307
Peptide Sequence
Calculated weight
Obtained Weight
N-terminal Sequencing
305 - 308:α 796.55 796.65 _DPR Cys 305:α
312 - 320:α 1442.4a 1748.4b
1441.52 1748.11
YMA_ _L Cys 315:α & Cys 316:α
340 - 352:α 1834.8c 1848.8d
1836.25 1848.05
SIQFV TIQFV Cys 347:α
351 - 359:β 1278.15 1278.36 TAV_D Cys 354:β
(B) Molecular mass of –CH2CONH2 = 58
Peptide Sequence
Calculated Weight
Obtained Weight
N-terminal Sequencing
305 - 308:α 547.55 546.7±0.1 _DPR Cys 305:α
340 - 352:α 1585.8c 1599.8d
-
TIQFV Cys 347:α
217 - 241:β 2710.05 2710.2±1.3 LTTPT Cys 239:β
351 - 359:β 1029.15 1029.11 TAV_D Cys 354:β
(C) Mass of (1) [14C]-NEM=127 and (2) [14C]-NEM+HOH=145
Peptide Sequence
Calculated Weight (e)
Obtained Weight
N-terminal Sequencing
305 – 308:α (1) 614.6 (2) 632.6
612.1 _DPR Cys 305:α
312 – 320:α (1) 1260.4 (2) 1278.4
1280.6
YMA_ _L Cys 315:α & Cys 316:α
340-352:α (1) 1654.80 (2) 1672.80
1671.6±1.5
TIQFV Cys 347:α
374-390:α
(1) 1935.12 (2) 1953.12
1934.02
AV_ML Cys 376:α
217 - 241:β
(1) 2779.05 (2) 2797.05
2798.4±0.8
LTTPT Cys 239:β
351 - 359:β
(1) 1096.15 (2) 1114.15
-
TAV_D Cys 354:β
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P. J. Britto, Leslie Knipling and J. WolffThe local electrostatic environment determines cysteine reactivity of tubulin
published online May 21, 2002J. Biol. Chem.
10.1074/jbc.M204263200Access the most updated version of this article at doi:
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