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 : janw@bdg8.niddk.nih.gov

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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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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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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Figure 6

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Figure 7

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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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