Eur. J. Biochem. 213, 321-328 (1993) 0 FEBS 1993

‘H-NMR spectroscopy of bovine lens P-crystallin The role of the PBZcrystallin C-terminal extension in aggregation

Philip G. COOPER, John A. CARVER and Roger J. W. TRUSCOTT Australian Cataract Research Foundation, Department of Chemistry, The University of Wollongong, Australia

(Received November 3, 1992) - EJB 92 1556

‘H-NMR spectroscopic studies of bovine eye lens P-crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three P-crystallin aggregates are dominated by resonances from the pB2 subunit, particularly from the N- and C-terminal extensions of this subunit. Resonances from other subunits, which all have terminal extensions, are, in general, absent from spectra of the P-crystallin aggregates. Therefore, the @2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other a-crystallin subunits. Furthermore, reso- nances arising from the C-terminal extension of pB2-crystallin are not present in the spectrum of the octomer, which is consistent with the C-terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C-terminal exten- sion of pB2 and the hydrophobic pBl subunit, which is only found in the octomer, is discussed. At higher temperatures (45OC) in the octomer, partial exposure of the C-terminal extension of pS2 occurs indicating that the octomer may be starting to break up into smaller aggregates.

The light refractive power of the vertebrate eye lens re- sults from the high concentration of soluble proteins, collec- tively referred to as crystallins. Some 40-48% of the crys- tallins in the bovine lens belong to the P-crystallin family [l, 21. The seven a-crystallin subunits have molecular masses ranging over 23-33 kDa [ 3 ] . As determined by size-ex- clusion chromatography, the P-crystallin subunits are aggre- gated species consisting of dimers, trimers and octomers, which are commonly referred to as pL2, pL, and PH respec- tively 14-61.

Based on their sequence similarity with the y-crystallins, it was proposed that each P-crystallin subunit consisted of four ‘Greek key’ motifs folded into two domains with the N- terminal globular domain of all P subunits having extensions of various lengths [7, 81. In addition, it was postulated that the three basic P subunits, pSl, pS2 and pB3, have exten- sions from the C-terminal domains [7,9]. Unlike the globular domains which are highly conserved across species, the ter- minal extensions of the P subunits display considerable se- quence variation across species, especially within the N-ter- minal extension [9]. It has been proposed that the N- and C- terminal extensions extend from the globular domains [8- 101. In support of this proposal, limited proteolysis of the @32 dimer results in preferential cleavage in the terminal extensions leaving the globular domains intact [ l l ] . The function of the terminal extensions is currently unknown, but it has been speculated that they act as attachment sites for oligomer formation or for binding to other cellular compo- nents [9].

Correspondence to J. A. Carver, Australian Cataract Research Foundation, Department of Chemistry, The University of Wollon- gong, Northfields Avenue, Wollongong NSW 2522, Australia

Fax: +61 42 214 287. Abbreviations. 2D, two-dimensional; TOCSY, total correlation

spectroscopy.

X-ray structural analysis of the predominant subunit, pB2-crystallin, confirmed the structural similarity of the 0- with the y-crystallins [12]. Low electron density was ob- served for the N- and C-terminal extensions in pB2, implying that these extensions move independently of the globular do- mains in the crystal state [12]. In agreement with the crystal structure, our ‘H-NMR studies in solution showed that the residues of the N- and C-terminal extensions in the pB2 di- mer give well resolved resonances, indicative of highly flex- ible extensions which adopt no ordered structure [13].

The present report is based on ‘H-NMR spectroscopy of the dimer, trimer and octomer P-crystallin aggregates. We find that resonances from the terminal extensions of pB2 dominate in the dimer and trimer spectra such that few reso- nances can be attributed to the terminal extensions of other /3 subunits. Thus, the terminal extensions of other P subunits do not have the solvent exposure and independent mobility of the pB2 terminal extensions. In the spectra of the octomer aggregate, resonances from only the N-terminal extension of pB2 are observed almost exclusively, implying that the C- terminal extension binds in the oligomer.

EXPERIMENTAL PROCEDURES Purification of /%crystallin

Bovine eyes were obtained from a local abattoir. Lenses were removed and the crystallins extracted in 50 mM Trisl HC1 pH 7.5, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM 1,4-dithiothreitol and 0.02% sodium azide (2 ml/lens) using a glass homogenizer. Insoluble material was removed by centrifuging the homogenate at 15000 g for 30 min at room temperature.

The crystallin separation method used was similar to that described by Kleiman et al. [14]. Initially, gel chromatogra-

322 Subunlt Dlstrlbutlon In the Heterodimer

I a I I

Fig. 1. The profile for /Jaystallin separation on a Sephacryl S- 300 column in 0.5 M sodium phosphate, 0.02 % sodium azide at pH6.8. The octomer, pH, elutes at approximately 200kDa, the trirner, el, at 70 kDa and the dirner, /?Lz, at 50 kDa. The horizontal bars indicate fractions collected for each aggregate.

165 190 215 240 255 270 285 ' Elution volume (ml)

phy on a column (0.05 X 0.5 m) of Sepharose CL-6B equili- brated in 50 mM Tris/HCl pH 6.8 and a flow rate of 16 ml/h was used to separate the soluble proteins into a-, P- and y- crystallin fractions. The P-crystallin fraction was concen- trated with a Diaflo ultrafiltration apparatus (Amicon) using a YMlO membrane. This fraction was then further separated into three size aggregates, PH, pL, and pL,, by chromatogra- phy using a column (0.02 X 1.00 m) of Sephacryl S-300 equilibrated in 50 mM sodium phosphate pH 6.8, 0.02% so- dium azide and a flow rate of 16 mlh. The three size frac- tions, PH, pL, and pL2, correspond to aggregate sizes of octo- mers, trimers and dimers respectively. Each fraction was dia- lysed against deionised water at 20 "C, lyophilised and stored at 4°C until prepared for 'H-NMR spectroscopy.

Determining the proportion of subunits in /I aggregates

Samples from each isolated aggregate were subjected to SDSPAGE electrophoresis on 13 -25 % gradient acrylamide gels in discontinuous buffer, similar to the method of Laemmli [15]. The proteins were stained with Coomassie blue R250 (Sigma). Four gels, each representing a different octomer CP,), trimer @L,) and dimer (BL,) aggregate separa- tion, were scanned using an LKB Ultrascan XL laser densi- tometer, and the relative absorption of the protein bands at 633 nm was determined. Areas under the peaks were meas- ured using the Gelscan XL software package to obtain the proportion of each P subunit present in the various aggre- gates.

Sample preparation for NMR spectroscopy

Generally, 2 -3 mM subunit concentrations of P-crys- tallins were prepared by dissolving between 50-70 mg of the lyophilised protein in 800 pl of 99.5 % D,O or 85 % H,O/ 15% D,O. An unlyophilised sample of the octomer was also prepared by dialysing to water, concentrating with a YMlO membrane and the addition of 15% (by vol.) D20 prior to NMR spectroscopy. 3-(Trimethylsilyl)-(2,2,3,3-2H,)propionic acid was used as an internal reference at 0 ppm.

'H-NMR spectra were acquired at 400 MHz and 23°C on a Varian Unity 400 NMR spectrometer. All two-dimen- sional (2D) spectra were recorded with the parameters and pulse sequences as described in the preceding paper [ 131. A

6 1 62 83 A1,2 ,4 A 3

8UbUnit

Subunlt Dlstrlbutlon In the Trlmer

B3 A1 ,2 ,4 A 3 B1 82

8ubUnlt

Subunlt Dlstrlbutlon In the Octomer

4 0 T ae Y

30 - I

c 3 20 a M

10

0 83 A 1 , 2 , 4 A 3 61 82

rubunlt

Fig.2. The proportion of subunits present in each p-crystallin aggregate as determined by integrating the area under the peaks using densitometric measurements of SDSEAGE gels. (a) Subu- nit proportions for the dimer, / j L z ; (b) subunit proportions for the trimer, pL,; (c) subunit proportions for the octomer, pH. Peaks re- sulting from minor protein bands are not included.

spin lock period of 45 ms was used for all total correlation spectroscopy (TOCSY) spectra.

RESULTS The separation of the P-crystallins using gel chromatog-

raphy on Sephacryl S-300 is shown in Fig. 1. Elution peaks at approximately 200, 70 and 50 kDa, corresponding to the octomer (pH), trimer W,) and dimer @L,) respectively, were isolated. Horizontal bars under the peaks show the fractions which were pooled for each aggregate. All aggregates were shown to consist of a range of P subunits as determined by

323

5. // H2Ol

v N15

4.0 3.5 3.0 2 .5 2 . 0 1.5 1.9

F1 (ppm)

Fig.3. A portion of the aliphatic region of a TOCSY spectrum of the dimer (BL,), in D,O, pH 4.8. The insert shows the H-2 to H-4 histidine aromatic cross-peaks. Annotated cross-peaks have previously been assigned to residues of the N- and C-terminal extensions of the pB2 homodimer [13]. The boxed cross-peaks are not observed in spectra of pB2-crystallin.

SDSPAGE. The relative concentrations of the p subunits within the three isolated p fractions were determined by SDS/ PAGE and densitometry. Fig. 2 shows the proportion of each p subunit in the dimer, trimer and octomer aggregates. The pB2 subunit predominates in each aggregate, representing about 45% in the dimer and 35% in both the trimer and octomer aggregates. The acidic p subunits, except for pA3, migrate together as a band corresponding to a molecular mass of 23 kDa, and therefore their proportions could not be determined separately.

The absence of pSl and PA3 subunits in the dimer (PL,) is consistent with the results of Slingsby and Bateman [16]. The pSl subunit is unique to the octomer [6]. pBl is re- solved into two bands, pSla and pBlb, on the SDSPAGE gels [17] and the results for these were combined in Fig. 2. For the trimer, the presence of 3% pBl is most likely due to cross contamination from the octomer fraction as these two size aggregates do not separate completely using size-ex- clusion chromatography (Fig. 1). Minor bands scanned in the gels are not included, hence the sum of those reported here do not add up to 100%.

These results were compared to an extrapolation of the subunit distribution in the p aggregates as determined by Slingsby and Bateman [16], who isolated the subunits from the different /3 aggregates by ion-exchange chromatography using fast protein liquid chromatography with a Mono-Q col- umn. Our results are in reasonable agreement with these au- thors except for the proportion of pA1, PA2 and pA4, for which we observed a higher percentage in all size aggregates (27%, 32% and 18% collectively in the dimer, trimer and octomer aggregates, respectively, compared to 15 %, 17 % and 12% by Slingsby and Bateman [16]). In our study of

pBZcrystallin [13], a different ion-exchange medium (Frac- togel DEAE) was used for chromatographic separation of the P-crystallins. Our experience suggests that the proportion of pA1, PA2 and PA4 subunits, as measured by gel scanning, over-estimates their relative concentrations which may be due to differential staining of the subunits by Coomassie blue. Having characterized the composition of the three p- crystallin aggregates, each fraction was subjected to two- dimensional (2D) 'H-NMR analysis.

Fig. 3 shows a portion of the aliphatic region of the TOCSY spectrum of the p dimer aggregate (PLJ in D,O. Most of the resonances observed correspond to residues of the N- and C-terminal extensions of the pB2 homodimer, which were previously assigned by us [13]. These resonances are labelled in Fig. 3 and the well resolved resonances that were not observed in the TOCSY spectrum of the pB2 homo- dimer are boxed. The insert shows the H-2 to H-4 cross- peaks for the histidine residues, three of which belong to the terminal extensions in the pB2 homodimer.

As was the case for the dimer, the N- and C-terminal extensions of the pB2 subunit are well resolved in the NMR spectrum of the trimer (Fig. 4). Despite the pB2 subunit rep- resenting little more than a third of the subunits in this aggre- gate (Fig. 2), there are fewer non-pB2 resonances observed in the trimer aggregate than in the dimer. The histidine H-2 to H-4 cross-peaks of pS2 are exclusively observed (see in- sert in Fig. 4). Interestingly, the cross-peaks for AsnlS, which is the residue present at the base of the N-terminal extension of pB2-crystallin, are more readily resolved in the trimer than in either the dimer (Fig. 3) or the pB2 homo- dimer [13].

324

7.48 7.30 7.20 7

N15

4.0 3.5 3.0 2.5 2.0 1.5 1.0

F1 lppn)

Fig.4. A portion of the aliphatic region of a TOCSY spectrum of the trimer a,), in D,O, pH 4.7. The insert shows the H-2 to H-4 histidine aromatic cross-peaks. Annotated cross-peaks have previously been assigned to residues of the N- and C-terminal extensions of the pS2 homodimer 1131. The boxed cross-peaks are not observed in spectra of the pB2 homodimer.

The TOCSY NH to a$ region for the trimer in H,O (Fig. 5) is almost identical to that obtained for the pB2 homo- dimer. The labelled residues correspond to that of the N- and C-terminal extensions of the pB2 subunit. As was found for the spectrum in D,O (Fig. 4), the cross-peak for Am15 in the trimer is resolved, whereas this resonance was not observed in the TOCSY spectrum of the pB2 homodimer. In Fig. 5, six cross-peaks are present for the NH to P-CH, cross-peaks of alanine residues. For the pB2 homodimer, eight NH to p- CH, resonances from all eight alanines in pB2-crystallin were observed [13]. Three of these resonances were assigned to Alal, Ala8 and Ala199 in the N- and C-terminal exten- sions with the remaining five unassigned resonances arising from alanine residues within the globular region of the pB2 subunit. The six alanine NH to P-CH, resonances observed in the trimer have identical chemical shifts to those in the pB2 homodimer spectrum [13]. Accordingly, Alal, Ala8 and Ala199 NH to p-CH, resonances are assigned in the trimer spectrum and the three unassigned NH to P-CH, resonances, labelled Aa, Ab and Ac in Fig. 5, arise from the pB2 subunits present in the trimer aggregate.

As was found for the dimer and trimer, in the octomer aggregate, most of the well resolved resonances arise from the pB2 subunit. In contrast to the trimer and dimer, however, the octomer aggregate gave rise to well resolved resonances only from the N-terminal extension of pB2. Fig. 6 shows a portion of the aliphatic region of the TOCSY spectrum in D,O. Absent in this spectrum are cross-peaks for the pB2 C- terminal residues : Ser204, Ser203, Pr0202, His201, Phe200, Ala199, Arg197, Gln196 and His195. The a-CH, resonances of Gly198 are equivalent and therefore have no cross-peaks

in this spectrum. Resonances from Gln193 and Trp194, al- though part of the C-terminal extension, were not observed in spectra of pB2-crystallin [ 131.

The NH to a,p region of a TOCSY spectrum of the oc- tomer in H,O also confirmed the absence of signals from the C-terminal extension of pB2 (Fig. 7). For example, compared to the trimer spectrum of Fig. 5, one less alanine NH to P- CH, cross-peak was observed which corresponds to the loss of the cross-peak for Ala199 of pB2.

There is some evidence to suggest that the quaternary structure of the octomer is temperature-dependent [l]. NMR studies on the octomer were therefore conducted at higher temperatures by acquiring TOCSY spectra at 37 "C and 45 "C. The TOCSY spectrum of the octomer in H,O at 37 "C showed no significant change in the number of resonances present, compared to those at 23"C, although resonances of the N-terminal extension were better resolved (spectrum not shown). For spectra acquired at 45 "C, however, resonances arising from Ser203 and Ser204 of the C-terminal extension of pB2 were observed (Fig. 8).

It has also been suggested that lyophilisation affects oc- tomer aggregation [ 1, 61. Accordingly, a TOCSY spectrum was acquired for the octomer which had not been lyophilised. The spectrum gave rise to resonances indistinguishable from those for lyophilised octomer in Figs 6 and 7. All the samples for NMR spectroscopy were freshly prepared from lyophi- lised protein aggregates. 1D spectra of the aggregates before and after acquisition of TOCSY spectra were identical, indi- cating that the aggregates were stable for the duration of the NMR experiments.

325

0 $- - US204

8.2-

8.4-

a.+

8.6- I

8.7-

I " " I " ' ~ I ~ ' " I " " l " " I " " I " " I ' ' '

F I Ippn)

5.0 4.5 4.e 3.5 3 . 0 8.5 2.8 1.5

Fig.5. The NH to a,B region of a TOCSY spectrum of the trimer (BL,), in H,O, pH 5.0. Annotated cross-peaks have previously been assigned to residues of the N- and C-terminal extensions of the pB2 homodimer [13]. With the exception of Asnl5, all resonances in this Fig. are observed in spectra of the pS2 homodimer.

:": 3.

3.

7.40 7.38 7.28 7.10 3.

-8 Pl3 u-4

a

I ~ " ' I " " I " ~ ' " " ' " ' ~ ~ I " ' ' I ' 4.8 3.5 3.0 2.5 2.8 1.5 1.8

F1 Iwm)

Fig. 6. A portion of the aliphatic region of a TOCSY spectrum of the octomer (pH), in D,O, pH 5.1. Cross-peaks that have previously been assigned to N-terminal residues of the /?J32 homodimer are indicated. Resonances from the C-terminal extension of gS2 are absent. Those not observed in the pB2 homodimer spectrum are boxed.

326

F2 . ( P r n

8.e-

8.1-

8.e-

8.3-

8 . e

8.5-

8.6-

8.7-

FE [yf

8.e-

8.1-

8.2-

8.3-

0.4-

8.5-

8.6-

8.7-

n

1 , , , , , , , , , , , , , , , . , , , , , , , , , , , , , , , , . , , 5.8 4.5 4.0 3.5 3.0 2.5 Z.O 1.5

F1 lppm)

Fig. 7. The NH to a,/l region of a TOCSY spectrum for the octomer (pH), in H,O, pH 5.1. Annotated cross-peaks have previously been assigned to residues of the N-terminal extension of the m 2 homodimer. No resonances for the C-terminal extension of pB2 are observed.

Q -

4 - - @ s o 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

F1 lppml

Fig. 8. The NH to a,/l region of a TOCSY spectrum of the octomer &), in H,O, pH 4.7, at 45°C. Cross-peaks from Ser203 and Ser204 are observed in addition to those present at 23°C (Fig. 7).

327

DISCUSSION

The dimer aggregates of the &crystallins consist of a mixture of the following subunit aggregates: pB2-pB2, pB2-

PA3 and pB1 subunits are not observed in the aggregates while PA4 probably occurs only as heterodimers [ 181. From densitometric measurements, pB2 was found to comprise some 50% of the subunits present in the dimer (Fig. 2). The TOCSY spectrum, however, of this dimer mixture showed resonances which arise almost exclusively from the residues of the N- and C-terminal extensions of the pB2 subunit [13]. The few well resolved resonances of the dimer that do not arise from the pB2 homodimer are boxed in Fig. 3.

X-ray crystal structure analysis of pB2-crystallin has shown that dimer formation occurs via the N- and C-terminal domains of one subunit interacting with the C- and N-ter- minal domains, respectively, of the other subunit [12]. The interface between the two domains is referred to as the PQ interface [12]. Given the high similarity (45-56%) of the globular domains of the B subunits [19], it seems likely that the PQ interface is the site of all heterodimer formation. Hence, one would expect to observe resonances from the terminal extensions of other /kxystallin subunits. However, even allowing for coincidence of the cross-peaks due to simi- larity between subunits and lower concentrations of some subunits, the absence of a significant number of resonances attributable to the terminal extensions of subunits other than pB2, indicates that these subunits have few, if any, flexible regions. Thus, the terminal extensions of the pB2 subunit appear to be unique in having conformational flexibility in the heterodimer. That pB2 may have significantly different structural properties compared with the other p subunits is supported by its unique capacity to denature reversibly at high temperatures [20, 211.

The trimer aggregate consists of about 35% of pB2 sub- units (Fig. 2). As illustrated by the TOCSY spectra of the trimer (Figs 4 and 5 ) , there are few resonances present that can not be attributed to the pB2 subunit, e.g. histidine H-2 to H-4 cross-peaks from the three histidine residues of the @32 terminal extensions are the only ones resolved in the trimer spectrum. In fact, the spectrum is essentially indis- tinguishable from that of a TOCSY spectrum of the pB2 homodimer [13]. An exception is the cross-peaks for Asnl5, which are clearly resolved, see Figs 4 and 5, in contrast to both the pB2 homodimer [13] and the heterodimer spectra (Fig. 3). From the crystal structure of the pB2 homodimer, Am15 is at the start of the PQ interface [12]. It would be anticipated, therefore, that Am15 would have restricted movement and thus lack the flexibility to be well resolved in the NMR spectrum. The well resolved resonances observed for Asnl5 in the trimer spectrum support the conclusions of Slingsby and Bateman [18] that the pB2-pA3 dimer is the major aggregated species in the trimer. As the PA3 subunit lacks a C-terminal extension, the PQ interface for the pB2- j?A3 dimer will not restrict Asnl5. Asnl5 will therefore have greater flexibility in the trimer compared to that in the dimer aggregate where pB2-pB2 subunit interaction predominates.

In the NH to a$ region of the trimer spectrum (Fig. 5), resonances arising from six of the eight alanine residues of @32 were observed, five of which are within the globular domains of pB2. Surprisingly, alanine cross-peaks for the pB3 and PA3 subunits, which comprise some 30% of the protein present in the trimer and have a total of 19 alanine residues, are not resolved in spectra of the trimer. Thus, de-

pB3, pB2-pA1, pB3-PA1, PAl-PAl and pB2-BA4 [3, 181.

spite the similarity of pB3 and PA3 with pB2, they have few, if any, flexible regions in the trimer aggregates. A similar conclusion can be drawn about the absence of resonances arising from pA1, PA2 and PA4 subunits. There is no in- crease in the number of resonances in the trimer spectrum over that observed for the pB2 homodimer spectrum. The unassigned resonances in the NH to a,/? region of the pB2 homodimer can be attributed to flexible regions in the con- necting peptide between the two domains (some tentative as- signments were made for this region [13]), and/or to reso- nances within the domains that give rise to well resolved resonances, as are observed for example, for the alanine resi- dues. Thus, in the trimer and heterodimer samples, the ter- tiary structure of pB2 appears to be less compact compared to the other subunits, i.e. it contains more flexible regions. The well resolved resonances of the N- and C-terminal exten- sions of pB2 in the trimer give a clear indication that these extensions are neither involved in, nor restricted by, aggre- gation to form the trimer.

The octomer is an aggregate of an estimated eight sub- units, giving a molecular mass of 150-210lcDa [6]. Fig. 2 indicates a pB2 subunit concentration of about 35% in this oligomer. In the TOCSY spectra for this aggregate (Figs6 and 7), resonances assigned to the C-terminal extension of pB2 are absent, i.e. the spectra contain resonances arising from only the N-terminal extension of the pB2 subunit. This is consistent with a loss of flexibility in the C-terminal exten- sion, suggesting that it is bound or else otherwise restricted in this aggregate with only the N-terminal extension of pB2 being exposed to solvent. Consistent with solvent accessi- bility of the N-terminal extension is the observation that the N-terminus is readily susceptible to proteolysis while part of the octomer [22]. With the exception of /3B1, the subunit composition in the octomer is similar to that in the trimer (Fig. 2). Since resonances from the C-terminal extension of pB2 are observed in the trimer, we conclude that different, specific, interactions between the domains of the trimer and the octomer subunits are unlikely to explain the loss of C- terminal resonances of pB2 in the octomer. Consequently, the C-terminal extension of pB2 is involved in large aggregate formation in the P-crystallin octomer.

As with the trimer, there appear to be no flexible regions in the subunits of the octomer other than those arising from the pB2 subunit. The pBl subunit has 57% similarity with pB2 [19] and represents 22% of the octomer aggregate (Fig. 2). The N-terminal extension of pBl is long (58 resi- dues) and contains 19 alanine and 18 proline residues, many of which alternate. No resonances were observed for these residues in the octomer spectrum. In fact, one less alanine (from Ala199 of pB2) is present in the spectrum of the oc- tomer compared to that of the trimer (Figs 7 and 5 , respec- tively). NMR studies on myosin light chain [23], which also contains multiple Ala-Pro residues, concluded that these alternating amino acids result in a trans configuration for the proline, leading to a stiffened rod-like structure. Reduced flexibility of the N-terminal extension in pBl would decrease the likelihood of its resonances being resolved.

The octomer tends to deaggregate when subjected to ex- tremes of pH, high sucrose concentration or high tempera- tures and also undergoes irreversible modification when ly- ophilised [ l , 61. In order to ensure that the differences ob- served between the TOCSY spectra of the octomer and the trimer and dimer were not an artefact of lyophilisation, a TOCSY spectrum of the octomer that had not been lyophi- lised was acquired. No changes were observed in the spec-

328

trum when compared to the spectrum of the octomer that had been lyophilised. Hence, it is concluded that any changes which occur to this aggregate as a result of lyophilisation are not observable by NMR spectroscopy, and therefore do not affect the N-terminal extension of pS2 in the octomer, nor the involvement of the C-terminal extension of P2 in oc- tomer aggregation.

The effect of temperature on the octomer aggregation was also investigated. The TOCSY specbum of the octomer at 37 "C was essentially the same as that at 23 "C, although the resonances were better resolved at the higher temperature. At 45 "C, the TOCSY spectrum revealed strong resonances originating from the last C-terminal residue, Ser204, and weaker resonances for Ser203 (Fig. 8). No other resonances attributable to the C-terminal extension were observed, i.e. only the last two C-terminal residues are exposed to solvent at this higher temperature. Li [ l] found, using size-exclusion chromatography at 45 "C, that the octomer deaggregates into dimers. It has also been reported that the octomer will tend to deaggregate at high dilutions 161. Our NMR studies were done at relatively high concentrations (=60 mg/ml), which favour octomer stability [6]. Under these conditions and at 45"C, the NMR data indicate that partial exposure of the C- terminus occurs in the octomer which may be associated with the octomer starting to break up into smaller units.

The octomer consists of all the p subunits found in the trimer plus the pBl subunit, which is required for octomer formation [3]. When the octomer is deaggregated in 1 M so- dium chloride, pB2, along with small amounts of other sub- units, are released from the aggregate [5]. Isolated pSl sub- units are insoluble in physiological buffer due to self-aggre- gation and precipitation [31. Hence, it is plausible that the pBl subunit forms a core of the octomer aggregate which is surrounded by more soluble trimer subunits. This interaction could be stabilized by the attachment of the pB2 C-terminal extension to the pBl core leaving the pB2 N-terminal exten- sion oriented into the solution. Hence, the C-terminal reso- nances of pB2 would not be observed in the NMR spectrum of the octomer. Alternatively, it is possible that the orien- tation of pB2 in the octomer aggregate is such that the C- terminal extension is restricted without actually binding. We are currently undertaking further studles to determine the role of the C-terminal extension of pB2 in octomer aggregation.

We thank Wellcome, Australia Pty Ltd for financial support. The purchase of the NMR spectrometer was partially funded by grants from The University of Wollongong, The Australian Research Coun- cil and The Ramaciotti Foundation.

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