Structural properties of frog muscle myosin

456

Biochimiea et Biophysica Acta, 537 ( 1 9 7 8 ) 4 5 6 - - 4 6 5 @) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 38052

T H R E E CLASSES OF S U L F H Y D R Y L G R O U P IN BOVINE ~-CRYSTALLIN ACCORDING TO REACTIVITY TO V A R I O U S REAGENTS

R O L A N D J. S I E Z E N *, F E R G.M. C O E N D E R S a n d H E R M A N J. H O E N D E R S

Department of Biochemistry, University of Nijmegen, Ni]megen (The Netherlands) ( R e c e i v e d April 1 4 t h , 1 9 7 8 )

Summary ~-Crystallin from calf eye lens is found to have 30 sulfhydryl groups per 800 000 daltons b y modification with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiopyridine in 6 M urea. These -SH groups can be divided into three different classes in native ~-crystallin from their reactivity with DTNB, 4,4'-dithiopyridine, iodoacetamide and ethylenimine. Results obtained with these reagents point to the presence of 7 + 1 thiol groups (Class I) which are likely to be surface exposed, with a concomitant 40--45% quenching of tryptophan fluorescence in DTNB-modified ~-crystallin. Another 10 _+ 1 thiol groups (Class II) must be in a h y d r o p h o b i c environment since they react only with the h y d r o p h o b i c reagents, causing a further decrease in fluorescence intensity. Class III, 13 + 2 thiol groups, is inaccesible to any of these reagents. Introduction of up to 15 negatively charged thionitrobenzoate groups or seven positively charged aminoethyl groups in the ~-crystallin molecule at pH 7.5--8.0 did n o t change the state of aggregation as judged from the sedimentation coefficients.

Introduction

~-Crystallin, the multi-subunit protein in the eye lens, is a microheterogeneous mixture of apparently spherical molecules with molecular weights ranging from 7 • 10 s to 1 0 . 1 0 s [1,2]. T w o main types of subunits occur in bovine ~-crystallin, the A2 and B2 polypeptide chains, which are 57% homologous [3,4] and are present in a ratio of approx. 3 : 1 [2,5]. * Present address: D e p a r t m e n t of Physical Biochemistry, The John Curtin School of Medical Research, The Ausixalian National University, Canberra City, A.C.T. 2601, Australia. Abbreviation: DTNB, 5,5'-dithiobis(2-nitrobenzoic acid).

457

The quaternary structure of ~-crystallin is unresolved, and it is of interest to know whether all subunits are in (semi-)equivalent positions such as in the spherical shells of viruses, or if there is an arrangement involving exposed and buried subunits. Chemical modification of -SH groups is a useful tool in this pursuit. The bovine A2 chain has only one cysteine residue, whereas B2 has none at all [3,4]. If all A2 chains are in equivalent positions, then all -SH groups should show the same reactivity towards~specific thiol reagents. In this paper we demonstrate that at least three classes of -SH groups occur with various reactivities towards DTNB, 4,4'-dithiopyridine, iodoacetamide and ethylenimine. Previous investigations of the reactivity of bovine ~-crystallin sulfhydryl groups with p-hydroxymercuribenzoate, N-ethylmaleimide and iodoacetic acid indicate that both buried and exposed sulfhydryl groups exist, the relative amounts depending on the isolation procedure of ~-crystallin [6--8]. In addition, all the sulfhydryl groups could be reversibly exposed if the pH was raised from 8.8 to 10.4, or the temperature increased from 34 to 70°C [6,7]. We will consider these findings together with our results in the discussion. Materials and Methods

Materials Bovine serum albumine, Sigma; Biogel A-5M, Biorad; DTNB, 4,4'-dithiopyridine, ethylenimine, iodoacetamide, urea p.a., Tris, EDTA, 1,4-dithioerythritol and phenylmethylsulfonylfluoride, all Merck. Iodoacetamide was recrystallized before use.

Preparation of ~-crystallin solutions The water-soluble lens proteins in a calf lens cortical extract were fractionated on a Biogel A-5M column [9], equilibrated with 20 mM Tris-HC1, 80 mM NaCl (buffer 1) containing 1 mM EDTA, 0.2 mM phenylmethylsulfonylflu0ride, pH 7.3 (20°C). The low molecular weight ~-crystallin was pooled, dialyzed against deionized water, lyophilized and stored at --20°C. Solutions of ~-crystallin were prepared in appropriate buffers and passed through Millipore filters (1.2 pm pore size) before use.

Modification of -SH groups DTNB. 0.5 ml 10 mM DTNB was added to 2.5 ml ~-crystallin solution (about 1 mg/ml} in buffer 1, with or w i t h o u t 6.6 M urea. The increase in absorbance at 412 nm was recorded at room temperature (20--25°C), relative to DTNB buffer control. The number of -SH groups modified was quantitated at pH 7.5, 8.0 or 8.6 using molar extinction coefficients of 13.9 • 103, 14.1 • 103 and 14.2 • 103 (extrapolated) M -1 • cm -~, respectively, for released thionitrobenzoate ion [10]. Samples of various degree of modification were dialyzed against buffer 1, concentrated by ultrafiltration and subjected to sedimentation analysis. 4,4'-Dithiopyridine. This reagent was dissolved in absolute ethanol to 2.7 M and then diluted with buffer to 10 mM. The thiol assay was performed as with DTNB in buffer 1 (pH 7.2) using a molar extinction coefficient of 1.98 • 104

458

M -1 • cm -1 for 4-thiopyridone at 324 nm [11]. Ethylenimine. Ethylenimine (1.5--30 pl) was added to 6.0 ml a-crystallin solution (about 1.6 mg/ml) in buffer 1 (pH 8.0 or 8.6) with or w i t h o u t 6.3 M urea. The reaction was stopped after 2 h at 20°C with a slight excess of 1,4-dithioerythritol. The aminoethylated a-crystallin sample was then dialyzed against buffer 1 (pH 8.0 or 8.6) to remove excess urea and/or reagents, and concentrated by ultrafiltration. Sedimentation analysis of some samples was performed at this stage. The number of -SH groups which had not been modified was determined by back-titration with DTNB in urea as described above. Iodoacetamide. 70 ~1 10 mM iodoacetamide was added to 6.0 ml a-crystallin solution (approx. 1.6 mg/ml) in buffer I (pH 8.0), with or w i t h o u t 6.3 M urea. After 30 min in the dark at room temperature excess urea and/or reagent was removed by gel filtration over Sephadex G-25 in the same buffer w i t h o u t urea. The carboxy-amidomethylated a-crystallin was concentrated by ultrafiltration and the number of unreacted -SH groups determined by back-titration with DTNB in urea as described above.

Fluorimetry Fluorescence emission spectra were recorded with a Perkin-Elmer fluorescence spectrophotometer MPF-4 coupled to a Perkin-Elmer model 56 recorder. Excitation was at 295 nm. The concentrations of native and DTNB-modified a-crystallin were all exactly 0.29 mg/ml in buffer 1 (pH 8.0).

Miscellaneous All Tris buffers were of ionic strength 0.1 at 20 mM Tris-HC1 and 80 mM NaC1 [12]. Protein concentrations were measured by the m e t h o d of Lowry et al. [13], using bovine serum albumin as standard. Ultrafiltration was done in a Minicon B15 (Amicon) apparatus. Absorbance was measured with a Beckman model 25 spectrophotometer and kinetics were monitored with a Beckman recorder. Sedimentation analysis was performed at 60 000 rev./min at 20°C in a Beckman Spinco model E ultracentrifuge using Schlieren optics. The A2 chain has 173 residues, one cysteine residue at position 131, one t r y p t o p h a n at position 9 and a molecular weight of 19.830 [3]. The B2 chain has 175 residues, two tryptophans at positions 9 and 60, and a molecular weight of 20 070 [4]. All calculations of the number of modified -SH groups are based on a molecular weight of 8{)0 000 for a-crystallin. Since the stoichiometry is (A)3B [2,5], a-crystallin of this molecular weight contains 30 A-chains and hence 30 -SH groups. The often cited A/B ratio of 2 : 1 is generally obtained as a stain ratio from polyacrylamide gels [1,9,14,15], but at least using Coomassie Brilliant Blue stain this converts to a 3 : 1 molar ratio taking into account that this dye (and others perhaps, also) binds only to amino groups [16]; this makes the a m o u n t of dye bound proportional to the number of lysine residues per polypeptide chain, which is 7 for A2 [3] and 10 for B2 [4]. Note that both molecular weight and subunit composition are average values for the ~-crystallin population [ 1,2], but heterogeneity is of minor importance in these modification studies.

459

Results DTNB

The reaction of a-crystallin -SH groups with an approx. 50-fold molar excess (over -SH groups) of DTNB at pH 7.5 is shown in Fig. la. An initial fast reaction in the first hour is followed by a much slower reaction which levels off after approx. 15 h at a total of 14.7 -SH groups modified. This total does not increase with a 100-fold molar excess of DTNB. Since the concentration of DTNB does not change appreciably during the reaction, the process can be treated as a pseudo first-order one. The reaction curve is transformed in Fig. l b according to the equation: log(SH= - - S H t ) = k ' t + log(SHoo) where S H = total modified -SH groups, S H t = modified -SH groups at time t, and k' = pseudo first-order rate constant [17]. The plot is biphasic, indicating the presence of two distinct classes of -SH group with different reactivities toward DTNB. The slow phase of the reaction gives a linear transform with k~ow = 0.036 rain-' and when extrapolated to t = 0 the number of slow-reacting -SH groups is found to be 10.4. The initial reaction is too fast for an accurate determination of the kfast but the number of fast-reacting -SH groups is 4.3 by subtraction (Table I). This experiment was repeated to test the reproducibility modifled

- SH

group5

30 O 20 eee

ee

10

l* • •

."

•

e ~

""

f 3020-

b

10864-

3-

2-

Fig. 1. K i n e t i c s of t h e m o d i f i c a t i o n o f ~ - c r y s t a l l i n w i t h a n a p p r o x . 5 0 - f o l d m o l a r e x c e s s of D T N B ( b u f f e r 1, p H 7.5; r o o m t e m p e r a t u r e ) (a) N u m b e r o f m o d i f i e d -SH g r o u p s / 8 0 0 0 0 0 d a l t o n s o f ~-eryst~llln vs. t i m e (h), c a l c u l a t e d f r o m t h e c o n c e n t r a t i o n of r e l e a s e d a n i o n m e a s u r e d a t 4 1 2 n m . ( b ) P s e u d o f i r s t - o r d e r p l o t o b t a i n e d f r o m (a) b y plotting log (SHoo - - S H t) o n the o r d i n a t e vs. t i m e . N o t e t h e change in the t i m e scale.

GROUPS

7.5 7.5

7.2

8.0

8.6 8.6 8.6 8.6 8.0 8.0 8.0

50 50

50

2

80 350 600 800 1100 1350 1600

DTNB

4,4'-Dithiopyridine

Iodoaeetamide *

1.6 1.6 1.6 1.6 1.6 1.6 1.6

1.6

0.8

0.8 0.8

Approx. protein concentration (mg/ml)

8.4 18.9 25.7 26.5 26.7 26.9 26.6

28.4

30.5

29.7 29.7

Total -SH in u r e a

120 120 120 120 120 120 120

30

300

900 1050

Reaction time (rain)

0 0.8 2.4 2.3 6.7 7.3 7.6

7.0

18.8

14.7 16.5

Total reacted -SH

Native a-crystallin

0 3 8 8 23 25 26

24

62

49 56

4.3 6.3

Fastreacting -SH

0.036 0.049

10.4 10.2

r

kslow ( r a i n -1)

Slowreacting -SH

g r o u p s t i t r a t a b l e w i t h D T N B in u r e a .

* T h e n u m b e r s o f -SH g r o u p s m o d i f i e d in t h e p r e s e n c e o r a b s e n c e o f u r e a , a n d t h e p e r c e n t m o d i f i e d -SH in n a t i v e ~ - c r y s t a l l i n , are b a s e d on a t o t a l o f 29.7 -SH

Ethylenimine *

pH

Approx. molar excess (×)

Reagent

T h e n u m b e r s o f m o d i f i e d - S H g r o u p s , b a s e d o n m o l e c u l a r w e i g h t o f 8 0 0 0 0 0 f o r c~-crystallin, are a c c u r a t e to +_3%.

MODIFICATION OF SULFHYDRYL

TABLE I

461 of the DTNB reaction and we found 6.3 fast-reacting and 10.2 slow-reacting -SH groups with k~ow = 0.049 min -1, in fair agreement with the first experiment (Table I). Under denaturing conditions, viz. 6 M urea, the reaction of -SH groups with DTNB is complete within minutes, and the total number of -SH groups is found to be 29.7 + 0.9, which is an average of several independent measurements (Table I). No change in sedimentation coefficient occurs after modification of either the fast- or slow-reacting -SH groups with DTNB at pH 7.5 (TabIe II).

4, 4'-Dithiopyridine Fig. 2 shows the kinetics of the reaction of ~-crystallin -SH groups with an approx. 50-fold molar excess of 4,4'-dithiopyridine at pH 7.2. Modification is faster and more extensive than with DTNB, viz. a total of 18.8 -SH groups reacts within approx. 4 h. The pseudo first-order plot is biphasic b u t the scatter of data points prevents an accurate quantitation of the fast- and slow-reacting -SH groups. In 6 M urea a total of 30.5 -SH groups could be modified with 4,4'-dithiopyridine (single determination).

Iodoacetamide With approx. 2-fold molar excess of iodoacetamide 7.0 -SH groups react within 30 min at pH 8.0 and this number does n o t increase after 60 min incubation. The low molar excess is recommended to avoid modification of residues other than cysteine [18]. In 6 M urea 23.6, 26.6 and 28.4 -SH groups reacted after 30, 60 and 120 min, respectively. The last value is slightly less than with DTNB or 4,4'-dithiopyridine in urea, b u t probably within experimental error (+3%) or possibly not even the final value considering the slow reaction with iodoacetamide in urea. The number of -8H groups modified with iodoacetamide was determined

T A B L E II S E D I M E N T A T I O N C O E F F I C I E N T S OF S U L F H Y D R Y L - M O D I F I E D ~ - C R Y S T A L L I N

Protein c o n c e n t r a t i o n 3 - - 4 r n g / m l in buffer 1. Reagent

Approx. molar excess

(x) DTNB

Ethylenimine

pH

Reaction time (h)

(Approx.) modified °SH groups

s20,w

0 5 10 15 15

17.9 * 1B.0

•

0 50 50 50 50

7.5 7.5 • 7.5 7.5 7.5

0 800 II00

8.6 8.6 B.O

* All s e d i m e n t a t i o n c o e f f i c i e n t s are +0.3 S.

48 1.5 4 24 48 2 2 2

0 2.3 6.7

17.7 17.5 17.5 17.2 17.5 17.0

462 r e l Q t ive

fluorescence

m o d i f i e d - SH g r o u p s 30-

20-

,~

_

,~

o o

g 0

10-:

. I

I

I

f

I

2

4

6

8

,

hr

J

300

r

,

320

r

i

340

,

r

360

,

nm

Fig. 2. K i n e t i c s of t h e m o d i f i c a t i o n of cz-crystallin w i t h a n a p p r o x . 5 0 - f o l d m o l a x excess of 4 , 4 ' - d i t h i o p y r i d i n e ( b u f f e r 1, p H 7.2; r o o m t e m p e r a t u r e ) . T h e n u m b e r of m o d i f i e d -SH g r o u p s / S 0 0 0 0 0 d a l t o n s of c~-crystanin w a s c a l c u l a t e d f r o m t h e c h a n g e in a b s o z b a n c e a t 3 2 4 n m [ 1 1 ] . Fig. 3. F l u o r e s c e n c e e m i s s i o n s p e c t r a of n a t i v e ( s p e c t r u m O) a n d D T N B - m o d i f i e d c~-crystaUin. T h e n u m bers 4.7, 5.3, 5.9, 7.1 a n d 10.6 r e l a t e t o t h e n u m b e r of m o d i f i e d -SH g r o u p s . P r o t e i n c o n c e n t r a t i o n is 0 . 2 9 m g / m l in b u f f e r 1, p H 8.0. T h e e x c i t a t i o n w a v e l e n g t h is 2 9 5 rim.

indirectly by 'back-titration' with DTNB in urea of unreacted groups, assuming a m a x i m u m of 29.7 titratable -SH groups (Table I).

Ethylenimine a-Crystallin was incubated for 2 h at 20°C and pH 8.0 or 8.6 with varying amounts of ethylenimine. The extent of aminoethylation of -SH groups in both native and denatured a-crystallin depends strongly on the molar excess of ethylenimine (Table I). At pH 8.0 maximally 7.6 -SH groups react in native a-crystallin and at pH 8.6 a limit of only 2.4 modified -SH groups is reached. However, it is still possible that more thiol groups will react with an even higher molar excess of ethylenimine. Reduction with 1,4-dithioerythritol prior to modification with ethylenimine does n o t affect the number of -SH groups which can be aminoethylated. Even in urea more than a 600-fold excess of ethylenimine is required to reach a limiting value of about 27 aminoethylated -SH groups (Table I). Apparently 3 -SH groups do n o t react at all with ethylenimine in the urea-denatured a-crystallin, b u t they can be subsequently titrated with DTNB. The degree of modification was always determined indirectly, as with iodoacetamide, by back-titration with DTNB. The sedimentation coefficient of ~-crystallin does n o t change upon aminoethylation of up to 7 -SH groups (Table II).

463

Tryptophan fluorescence The fluorescence of tryptophanyl residues in a protein is sensitive to local variations in tertiary structure, for instance due to chemical modification of neighbouring residues [ 19]. The fluorescence emission spectrum of native a-crystallin (Fig. 3, spectrum 0) shows a prominent peak at 332 nm, when excited at 295 nm, which is mainly attributable to tryptophan residues [19]. ~-Crystallin modified with DTNB also has an emission maximum near 332 nm, b u t the intensity is lower and depends on the number of -SH groups modified. At least two stages are discernible, viz. with 5--7 groups modified the intensity is 40--45% lower, and when more than 10 -SH groups have reacted a 60% reduction of fluorescence intensity is found relative to native a-crystallin (Fig. 3). Discussion As theoretically predicted, a total number of 30 -SH groups/800 000 daltons of ~-crystallin is indeed found, by modification with DTNB and 4,4'-dithiopyridine in urea (Table I), confirming the (A)3B stoichiometry. We propose that these 30 -SH groups can be divided into three different classes in native ~-crystallin from their reactivity with various -SH reagents (Table III). Class I would consist of some 7 ± 1 thiol groups (23 + 3%) which are probably exposed on the surface of the a-crystallin molecule and react with all thiol reagents, albeit at different rates depending on size, charge or hydrophobic/ hydrophilic nature of the reagent. Note that we have n o t proven that the same 7 + 1 -SH groups are modified with each reagent, b u t we assume this to be the case. The 40--45% quenching of tryptophan fluorescence accompanying the modification of Class I -SH groups suggests that t r y p t o p h a n y l residues are in the vicinity of these cysteinyl residues. The sole tryptophan in the A2 chain at position 9 is a likely candidate since our limited proteolysis studies indicate that this tryptophan is exposed at the surface in native ~-crystallin, being accessible to chymotrypsin [20]. Class II comprises approx. 10 + 1 thiol groups (33 + 3%) which must be in a very hydrophobic environment, because they react only with the large, hydroTABLE III CLASSIFICATION OF SULFHYDRYL Reagent

GROUPS IN ~-CRYSTALLIN

Introduced

Class

charge

DTNB 4,4'-Dithiopyridine P-Hy droxymercurlbenzoate *

Iodoacetamide Ethylenimine * A d a p t e d f r o m r e f . 6.

negative none negative none positive

I

II

Surface exposed

Hydrophobic pocket

4--7 <' < 7 7--8

18--19 .16

10--11 ~ ~ 4 <'

III Buried

13--15 11--12 14 23 22--23

>