Variants of b 2 -microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature Maria C. Mimmi 1 , Thomas J. D. Jørgensen 2 , Fabio Pettirossi 1 , Alessandra Corazza 1 , Paolo Viglino 1 , Gennaro Esposito 1 , Ersilia De Lorenzi 3 , Sofia Giorgetti 4 , Mette Pries 5 , Dorthe B. Corlin 6 , Mogens H. Nissen 5 and Niels H. H. Heegaard 6 1 Dipartimento di Scienze e Tecnologie Biomediche and MATI Centre of Excellence, University of Udine, Italy 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark 3 Department of Pharmaceutical Chemistry, School of Pharmacy, University of Pavia, Italy 4 Department of Biochemistry, School of Pharmacy, University of Pavia, and Biotechnology Laboratories, IRCCS Policlinico San Matteo, Pavia, Italy 5 Institute of Medical Anatomy, University of Copenhagen, Denmark 6 Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark Keywords amyloidosis; cleaved b 2 -microglobulin; human b 2 -microglobulin; NMR; protein conformation Correspondence N. Heegaard, Department of Autoimmunology, Statens Serum Institut 81 ⁄ 536, Artillerivej 5, DK-2300 Copenhagen S, Denmark Fax: +45 32683876 Tel: +45 32683378 E-mail: nhe@ssi.dk (Received 31 January 2006, accepted 31 March 2006) doi:10.1111/j.1742-4658.2006.05254.x Cleavage of the small amyloidogenic protein b 2 -microglobulin after lysine- 58 renders it more prone to unfolding and aggregation. This is important for dialysis-related b 2 -microglobulin amyloidosis, since elevated levels of cleaved b 2 -microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved b 2 -microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine b 2 -microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of b 2 -microglobulin from which lysine-58 is removed, is much reduced com- pared to wild-type b 2 -microglobulin and is strongly dependent on tem- perature and protein concentration. 1 H-NMR spectroscopy and amide hydrogen ( 1 H ⁄ 2 H) exchange monitored by MS show that the overall three- dimensional structure of the variants is similar to that of wild-type b 2 -microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E b-strands close to the D–E loop cleavage site at lysine-58, and the experiments suggest con- formational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered b 2 -microglo- bulin molecules. This is the first single-residue resolution study of a specific b 2 -microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in b 2 -microglobulin amyloidogenicity in vivo. Abbreviations b2m, b 2 -microglobulin; CE, capillary eletrophoresis; cK58-b2m, b 2 -microglobulin cleaved after lysine-58; dK58-b2m, b 2 -microglobulin with lysine-58 deleted; DRA, dialysis-related amyloidosis; DN3-b2m, b 2 -microglobulin devoid of N-terminal tripeptide; FID, free induction decay. FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2461 The conformational behavior of b 2 -microglobulin (b2m) is of interest because this molecule is involved in dialy- sis-related amyloidosis (DRA) [1,2]. This condition, somehow induced by long-standing dialysis or renal insufficiency, is characterized by fibrillation and precipi- tation of b2m in osteoarticular tissues. Under normal conditions, b2m is a soluble plasma protein and also part of the MHC class I complexes on the surface of nucleated cells. It has become clear that this compact, seven b-stranded protein is conformationally unstable after cleavages and truncations, and that even intact b2m may, to a minor extent, adopt an alternative con- formation at physiological pH [3]. Amyloid fibril forma- tion from b2m in vitro requires nonphysiological conditions with respect to pH and ionic strength, the presence of divalent metal ions, or some of the trunca- tions ⁄ deletions that have been reported to be present in b2m extracted from amyloid lesions [4–6]. The study of the behavior of b2m and b2m variants is relevant not only for DRA, but also for understanding common pathways of fibril formation in amyloidotic conditions such as Alzheimer’s disease, transthyretin amyloidoses, immunoglobulin fragment amyloidosis, or some of the many other types of amyloidoses [7]. We have previously characterized two b2m variants, the first obtained by cleavage after Lys58 (cK58-b2m), and the second by further deletion of the same residue (dK58-b2m) (Fig. 1). It was shown that the concerted action of activated complement C1s and carboxypepti- dase B cleaves b2m after Lys58, leading to cK58-b2m, and removes the same residue to generate dK58-b2m [8]. This limited proteolysis attacking a susceptible peptide bond residing in the loop between b-strands D and E of b2m (Fig. 1) increases the conformational heterogeneity of the cleaved b2m compared with the wild-type (wt) molecule [9,10]. The dK58-b2m variant may occur in vivo and has been reported to be gener- ated in sera from patients with inflammation patho- logies, cancer, and renal insufficiency [11–13]. Additionally, we recently showed, using dK58-b2m- specific antibodies, that dK58-b2m circulates in the blood of many dialysis patients [14]. The conformations of cK58-b2m and dK58-b2m have not previously been probed at the single amino acid level and correlated with the solution stability of these molecules. We therefore here explore the struc- tural features and stability of the Lys58-cleaved b2m variants compared with those of wt b2m by a Lys58 wt-β 2 m cK58-β 2 m dK58-β 2 m A B C a b 1 SS 99 581 S S 59 1 S S 9959 c 99 57 Lys 58 + Fig. 1. Structures of b 2 -microglobulin (b 2 m) and b 2 m variants. (A) View of the 20 best-fitting solution structures of wild-type b2m based on NMR restraints and tethered molecular dynamics. For the sake of simplicity, only the backbone is drawn, apart from the side chain of Lys58, which is highlighted. Designation of b-strands A–G is indicated. The local trace thickness corresponds to the spatial spreading over the best overlap of the structural family ensemble. Only the first members of the solution structure families were considered. Drawn with MOLMOL [34]. (B) NMR-based solution structure of monomeric b2m (pdb entry: 1JNJ) in a ribbon drawing. The Lys58 residue (in red) and the Cys25 and Cys80 residues (yellow) connected by a disulfide bridge are shown in the backbone trace. Drawn with WEBLABVIEWERPRO 3.7. (C) Schematic drawing of the variants of b 2m generated by limited proteolysis of the wild-type molecule. From the single-chain wild type, a heterodimeric molecule (cK58-b2m), in which the two chains are connected by a disulfide bridge, is generated by cleavage between the Cys residues. The further trimming (removal of Lys58) of cK58-b2m generates the dK58-b2m variant. Stability of b 2 -microglobulin cleaved at Lys58 M. C. Mimmi et al. 2462 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS combination of NMR spectroscopy, MS, and capillary electrophoresis (CE). Results and Discussion Solution stability of cleaved b2m variants monitored by 1 H-NMR spectroscopy When b2m is modified by limited proteolysis cleaving the chain between the Cys25 and Cys80 residues, a heterodimeric molecule consisting of two chains held together by a disulfide bridge is generated. This mole- cule (cK58- b2m) is further processed in vivo to the dK58-b2m variant, which lacks the K58 residue exposed in the A-chain of cK58-b2m (Fig. 1) [11]. The behavior of cK58-b2m and dK58-b2m in solution was studied by a series of one-dimensional 1 H-NMR spec- tra collected at different conditions of temperature and protein concentration. The stability of concentrated solutions (c. 0.3 m m) in the temperature range between 288 and 310 K was first investigated. The one-dimen- sional 1 H spectra of cK58-b2m and dK58-b2m collec- ted at 288 K (Fig. 2A) exhibit the typical resonance pattern of the folded protein, with a few resolved peaks in the aliphatic and aromatic regions. In partic- ular, the upfield shifts of Val37, Ile35 and Leu23, due to the proximity of aromatic residues such as Tyr66, Phe30, Phe70 and Trp95 (Fig. 2B), are diagnostic of tertiary structure interactions in the hydrophobic core and represent a signature of the native fold of the b2m molecule (Fig. 2A, lower panel) [15]. When the tem- perature is increased in steps of five degrees up to 298 K, the lower solution stability of dK58-b2m com- pared to cK58-b2m is highlighted. While the latter at 298 K maintains a folded conformation, the variant devoid of Lys58 is less stable and undergoes slow unfolding and aggregation over time, as shown in Fig. 3. The unfolding is evidenced by the progressive loss of spectral spreading and the simultaneous growth of some main envelope at the typical frequencies of unfolded polypeptides (around 1 p.p.m.). The format- 78910 p.p.m. 2 1 0 p.p.m. cK58 288K dK58 288K L23F70 A V37 L23 L23 L23 V37 I35 I35 F70 78910 p.p.m. wild-type 310K 2 1 0 p.p.m. V37 L23 I35 L23 F70 B Fig. 2. (A) One-dimensional 1 H-NMR aliphatic (right) and aromatic (left) region of b 2 -microglobulin (b2m) cleaved after Lys58 (cK58- b2m) and b2m with Lys58 deleted (dK58-b2m), 0.3 m M,at288Kand pH 7.4, and of wild-type b2m, 0.7 m M, at 310 K at pH 6.6, observed at 500 MHz. The upfield shift of Val37, Ile35 and Leu23, which is diagnostic of tertiary structure interactions in b2m native folding, is highlighted. (B) Representation of the b2m hydrophobic core and of the aliphatic residues giving rise to the most upfield-shifted methyls in the 1 H-NMR spectrum. Val37, Ile35 and Leu23 (green) are placed in the shielding cone of aromatic rings (red). Only the most important residues are included in the plot. The plot was drawn using WEBLAB- VIEWERPRO 3.7 (Accelrys Inc., San Diego, CA, USA). M. C. Mimmi et al. Stability of b 2 -microglobulin cleaved at Lys58 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2463 ion of large aggregates is suggested by the broadening linewidth and the related decrease of the overall integ- ral value under equivalent NMR acquisition condi- tions. Over the )2 ⁄ 12 p.p.m. region, the spectra of dK58-b2m shown in Fig. 3 exhibit signal losses of 16% and 33%, respectively, corresponding to 10 and 41 h at 298 K. In the absence of overt precipitation, this suggests the formation of aggregates with substan- tially larger linewidths. The loss of stability and the formation of large, soluble aggregates in dK58-b2m solutions at 310 K over time were suggested previously by CE analyses, and evidenced by size-exclusion chromatography with light-scattering detection. In these experiments a well-defined aggregate formation with an aggregate size of about 50 nm or 5 · 10 6 gÆmol )1 was noted [10]. No estimate of the aggregate dimensions by measurement of translational diffusion coefficients using diffusion-ordered 2D-NMR spectroscopy experiments [16,17] was possible in the present study, because the relatively low sample con- centration (0.3 mm) prevented reliable exponential fit- ting of the experimental data. Upon further increase of the temperature to 310 K, cK58-b2m eventually slowly undergoes the same unfolding–aggregation process as observed for dK58- b2m (data not shown). In accordance with earlier observations using other methods [10], this thermal transition is irreversible (data not shown). Fig. 3. One-dimensional 1 H-NMR traces of b 2 -microglobulin (b2m) cleaved after Lys58 (cK58-b2m) and b2m with Lys58 deleted (dK58-b2m) at 298 K and pH 7.4. At 298 K, cK58-b2m exhibits the typical folded protein spectrum, whereas dK58-b2m undergoes an unfolding–aggregation process that is monit- ored at 0, 10 and 41 h from the temperature setting. The intensity of the upfield-shifted resonances of Leu23, Ile35 and Val37 gradu- ally diminishes, while the envelope around 1 p.p.m. increases. Simultaneous changes are observed in the aromatic region, invol- ving a loss of signal dispersion. The overall integral value is reduced after 41 h. Stability of b 2 -microglobulin cleaved at Lys58 M. C. Mimmi et al. 2464 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS A different behavior is found when obtaining a ser- ies of one-dimensional 1 H spectra at 310 K using more dilute solutions of cleaved b2m variants (c . 0.05 mm). In contrast to the results at 0.3 mm, the unfolding– aggregation process at a concentration of 0.05 mm is very slow. This is indicated by only a minor decrease of the diagnostic upfield-shifted peaks of Leu40, Val37, Ile35 and Leu23, even after 4 days (Fig. 4). Nevertheless, a slight and continuous modification of the tertiary structure is evident from the slow overall drift of the resonance system with a pattern suggesting loss of conformational homogeneity. After some 60 h, for both cK58-b2m and dK58-b2m, the presence of shoulders within the monitored isolated peaks indicates the presence of two or more conformers in equilibrium (peak shoulders are indicated by asterisks in Fig. 4). Further evidence for conformational heterogeneity comes from several other envelope changes that appear when the spectra are superimposed (data not shown). Protein aggregation monitored by capillary electrophoresis In contrast to wt b2m, which is freely soluble in physiological buffers up to at least 10 mgÆmL )1 (0.85 mm), the cleaved variants, in particular dK58- b2m, are prone to aggregation at high protein concentrations, especially at increased temperatures. Visible precipitation occurs over time at concentra- tions higher than 2 mg mL )1 (0.17 mm) for the dK58 variant; the cK58 variant is more stable. The aggregation behavior at different concentrations and temperatures was characterized by CE (Fig. 5). In these experiments, the changes in the amount of sol- uble material were followed over time. As shown in Fig. 5A, a 1 mgÆmL )1 (0.09 mm) dK58-b2m solution incubated at increasing temperature initially exhibits a shift in the conformational equilibrium between the fast (f) and slow (s) species to more of the (s) species, which is believed to be a partly unfolded intermediate (as can be seen below). Subsequently, at higher temperatures, an irreversible loss of soluble material occurs. In Fig. 5B, an analysis of soluble material over time at a fixed sample temperature of 308 K at two different protein concentrations, 0.9 mgÆmL )1 (0.08 mm) and 2.5 mgÆmL )1 (0.22 mm), clearly show the loss of solubility in the higher- concentration solutions of both variants, whereas at lower concentrations both species have constant peak areas from 0 to 24 h. This dependence of the Fig. 4. Details of one-dimensional 1 H-NMR traces of diluted b 2 -microglobulin (b2m) with Lys58 deleted (dK58-b2m) and b2m cleaved after Lys58 (cK58-b2m) solutions (0.05 m M) at 310 K and pH 7.4. At low concentration, the unfolding process is very slow, as indicated by an only very minor decrease of the intensity of the diagnostic peaks of Leu23, Ile35, Val37 and Leu40, even after some days. The presence of one or more conformational isomers, indicated by the resonance splitting of some isolated peaks (highlighted by asterisks), is particularly manifest in the spectra recorded after more than 100 h of incubation at 310 K, but may also be noticed after 60 h and to some degree in the very first recorded spectra (t ¼ 0 h). The increasing splitting between the peaks assigned to Ile35 H d1 and Leu23 H d2 , which is especially noticeable in the left panel, is consistent with a slow, continuous modification of tertiary structure, which takes place at 310 K in the dilute protein solution. M. C. Mimmi et al. Stability of b 2 -microglobulin cleaved at Lys58 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2465 solution stability of cleaved b2m on its concentration is in agreement with the NMR results presented above. MS analysis of global conformation by amide hydrogen ( 1 H ⁄ 2 H) exchange We have previously shown that native wt b2m and dK58-b2m undergo transient cooperative unfolding, evidenced by a correlated isotopic exchange of amide hydrogens [10]. This type of exchange mechanism (EX1) leads to the appearance of distinct bimodal isotopic envelopes in the mass spectra. The lower mass peak of this envelope represents the population of mol- ecules that has not yet undergone cooperative unfold- ing; the higher mass peak represents the population of molecules that has been in the unfolded state and thus undergone correlated exchange. To investigate the structural stability of the folded states of wt b2m, cK58-b2m and dK58-b2m, the exchange kinetics of the folded populations were determined at 298 K (Fig. 6). At this temperature, a gradual mass increase with exchange time is observed for the lower-mass popula- tion. This is due to the noncorrelated exchange mech- anism, which in structural terms can be explained by small-amplitude fluctuations within the protected core. The noncorrelated isotopic exchange kinetics shown in Fig. 7 was determined by the mass difference of the lower-mass populations relative to the fully deuterated control. Fig. 7 shows that at the shortest deuteration period (t ¼ 0.5 min), all three proteins contain the same number (i.e. 32; this number is also displayed in Fig. 6) of 1 H atoms not yet exchanged for deuterium. This indicates that an identical number of protecting hydro- gen bonds exists in the folded states of wt b2m, cK58- b2m and dK58-b2m. Furthermore, the cleaved variants, cK58-b2m and dK58-b2m, exhibit very similar noncor- related exchange kinetics (Fig. 7). This indicates that the stability of the hydrogen bond network that confers protection against isotopic exchange is almost identical for these proteins. However, with prolonged incubation this network appears to be slightly more stable in wt b2m than in the cleaved species (Fig. 7). Thus, in accordance with the NMR results, the glo- bal conformation of wt b2m appears to be conserved in the cleaved forms of b2m. Note that in these experi- ments only the slowly exchanging hydrogens are monitored. Thus, contributions from amide hydrogens in loop regions and from new termini generated in the cleaved variants are not expected to affect the exchange count. Two-dimensional NMR characterization The detailed interpretation of the 1 H-NMR spectra of b2m variants is based on the parent spectra of wt b2m obtained at different temperature and pH values 0 200 400 600 800 1000 1200 1400 1600 0 20 40 60 80 100 0.22 mM dK58-β2m 0.08 mM dK58-β2m 0.08 mM cK58-β2m 0.21 mM cK58-β2m Incubation time (min) at 35°C % [P/M]/[P/M] start B 8.5 9.0 -0.005 0.000 0.005 0.010 0.015 0.020 0.025 Time (min) A 200 nm 51.8°C 49.6°C 45.7°C 41.5°C 34.0°C 22.6°C 9.7°C f s A Fig. 5. Capillary electrophoresis separation of b 2 -microglobulin (b2m) with Lys58 deleted (dK58-b2m) incubated at different tempera- tures. (A) Separation profiles to show that sample temperature (indicated in the figure) influences the ratio between f and s con- formers of dK58-b2m in CE. All CE experiments were performed at a constant capillary temperature of 278 K to preserve the dis- tribution of conformers in the injected samples. Shown are over- layed electropherograms with time windows showing the s and f conformer peaks. Samples were 1 mgÆmL )1 dK58-b2m electro- phoresed at 278 K using 90 lA constant current after injection for 2 s. (B) Aggregation propensity of b2m cleaved after Lys58 (cK58-b2m) and dK58-b2m at different protein concentrations. Sol- uble material was monitored by CE as a function of incubation time. Samples of 0.9 mgÆmL )1 (triangles) or 2.5 mgÆmL )1 (circles) b2m variants (cK58, open symbols; dK58, filled symbols) were kept at 308 K, and aliquots (2 s injections of high-concentration samples and 4 s injections of low-concentration samples) were analyzed by CE performed at constant current of 80 lA, with the capillary cooling fluid maintained at 278 K. Samples also contained 0.2 mgÆmL )1 of a marker peptide. Shown are the summed peak areas P (total area of f + s peaks) divided by the marker peak area M at different time points as a percentage of the initial value of P ⁄ M at the onset of the experiments where the sample temperature was brought from 278 K to 308 K. Stability of b 2 -microglobulin cleaved at Lys58 M. C. Mimmi et al. 2466 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS [15,18] and requires the checking and redetermination of most of the resonance assignments of the molecule under investigation according to the standard meth- odology, i.e. going through scalar and dipolar connec- tivity patterns for each amino acid residue [19]. This work could be almost entirely completed for cK58- b2m, but only partially for dK58-b2m. The difficulty with both variants, particularly dK58-b2m, is due to their thermal lability (unfolding and aggregation). This prevented the use of optimal temperatures (e.g. 310 K) to improve data quality with concentrated samples (e.g. 0.5 mm). Increasing the temperature up to 320 K, whenever possible, generally improves the NMR data quality for 10–15 kDa proteins by reducing linewidths and thus favoring spectral analysis. As a compromise in the present study, the two-dimensional TOCSY and NOESY spectra of cK58-b2m were obtained at 298 K, whereas the best results with dK58-b2m were generated at 310 K by working with a very dilute sample (0.05 mm). The assignment lists (supplemental Tables 1 and 2) indicate an overall conservation of the resonance fre- quencies with respect to the corresponding wt values and thus confirm the retention of the main features of the native structure in both variants. The backbone H a chemical shifts of cK58-b2m and dK58-b2m (wherever assignments were available) were compared to the corresponding values of the wt protein, as shown in Fig. 8. As expected, the largest deviations of H a chem- ical shifts of cK58-b2m are found in proximity to the cleavage site, more specifically in fragments 56–58 and 59–64, i.e. at the opened loop D–E, and at the start of strand E [15]. Interestingly, similar deviations are also found in fragment 26–35, i.e. at the end of strand B and at loop B–C, which faces the D–E region. Accord- ing to the well-established correlation between H a chemical shifts and secondary structure in polypeptides [20], the shifts of cK58-b2m H a resonances compared to wt b2m reflect changes in the backbone arrangement within the D–E as well as in the B–C loop. Thus, two 1930 1950 1970 1990 m/z 11600 11700 11800 11900 Da Relative Abundance Min 0 0.5 dK58, 9+ 30 80 160 100% D ox ox 54 Da dK58 ox ox 32 Da 3255 cK58 cK58, 9+ Fig. 6. Global amide hydrogen ( 1 H ⁄ 2 H) exchange analysis of the folded conforma- tions of b 2 -microglobulin (b2m) cleaved after Lys58 (cK58-b2m) and b2m with Lys58 dele- ted (dK58-b2m) at 298 K in deuterated NaCl ⁄ P i . The proteins were incubated pairwise in deuterated NaCl ⁄ P i buffer. After various periods of deuteration, isotopic exchange was quenched by acidification. Subsequently, the samples were desalted at quench conditions and analyzed by ESI-MS. Shown are the ESI mass spectra of a mix- ture of cK58-b2m and dK58-b2m obtained after various deuteration periods (given in minutes in the figure) at 298 K. Left panel: deconvoluted ESI mass spectra. Right panel: ESI mass spectra of the m ⁄ z region with the [M + 9H] 9+ ions. The spectra obtained at t ¼ 0 min (i.e. lowest traces) were obtained from 1 H 2 O. Ox, Met99- oxidized species. M. C. Mimmi et al. Stability of b 2 -microglobulin cleaved at Lys58 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2467 opposite changes of secondary structure are found at the end of strand D and the beginning of strand E, i.e. a further loss in D and a stabilization in E of the local b-structure geometry. Compared with wt b2m, the cK58-b2m molecule is thus most conformationally different in the cleavage site region (D–E loop), with additional involvement of the adjacent residues of strands D and E, and the facing residues of loop B–C. Unfortunately, this analysis could not be extended to dK58-b2m, because of the ambiguous assignment of residues from these regions of the molecule. Conformational heterogeneity of cK58-b2m and dK58-b2m The whole NMR dataset for cK58-b2m and dK58- b2m revealed the occurrence of at least two different conformers for each molecule. These conformers were undergoing slow exchange on the chemical shift time- scale. Examination of the two-dimensional maps obtained with concentrated cK58-b2m at 298 K showed a generalized resonance doubling at the loca- tions and to the extent reported in Fig. 9. The features of the pattern of the second conformer resemble the features of a minor monomeric intermediate occurring along the b2m-refolding pathway that was named I 2 and initially identified by Chiti et al. in wt b2m [3]. The I 2 conformer was subsequently also detected in real-time NMR experiments [21]. Further analysis of other amyloidogenic b2m variants, and in particular of the species devoid of the N-terminal tripeptide, DN3-b2m, has shown that the I 2 conformer is in equi- librium with the fully folded species [21,22]. This indi- cates that it can be precisely identified through NMR characterization. In the case of DN3-b2m, the observa- tion of resonance doubling for the side chain signals of residues Val9, Ser11, Leu23, Val37 and Ala79, which are all close to one or more aromatic residues in the cluster of Tyr26, Tyr66, Phe70, Tyr78 and Trp95 [15], strongly suggested that I 2 corresponds to a slightly destabilized fold that has the overall conformation of wt b2m, but exhibits a looser packing of its hydropho- bic core. This interpretation was recently challenged by Kameda et al. [23], who reported evidence in favor of a slow trans–cis isomerization of Pro32 during refold- ing of b2m. Whatever the origin of the conformational equilibrium that gives rise to the slow refolding step of b2m, the proposed correspondence of the second form observed in the cK58-b2m spectra with the I 2 con- former identified in DN3-b2m is based on the similar- ity of the resonance doubling patterns of the two variants. This analogy is visualized in Fig. 10, where details of NOESY spectra are shown. In spite of the different conditions of temperature and pH, the close similarity of the patterns is readily appreciated. The excellent resolution of the resonances in Fig. 10 could not be exploited for quantitation of the relative con- centrations of the two forms because, in general, NOESY cross-peak amplitudes are determined by the actual motional characteristics of the connected nuc- lear pairs, and thus may differ between distinct con- formers [24]. Many other resonance doublings were observed (the most relevant are reported in Fig. 9), all consistent with the expected pattern of the I 2 interme- diate that was unambiguously recognized in previous studies of other b2m variants [21,22]. The best estimate of the equilibrium populations of the fully folded and I 2 forms for cK58-b2m at 298 K was obtained by using, for each conformer, the pair of TOCSY connec- tivities assigned to Val37 H c1 –H c2 . Taking into account the partial overlap of the specific cross-peaks, the resulting relative amount of I 2 at 298 K was 19 ± 9% of the total protein. The occurrence of an I 2 intermediate in equilibrium with the main species was also deduced from the dK58-b2m NMR spectra, although the lower resolution made it necessary to rely more on peak shape distortion than on actual peak separation (Figs 4 and 11B). Two conformers of the b2m variants cleaved at Lys58 were also detected by CE as previously reported [9] and are shown in Figs 5A and 11A. The precise nat- ure of the slow conformer peak (labelled ‘s’ in Fig. 5A and 11A) could not be unequivocally determined in these experiments. The two populations observed in CE 0 5 10 15 20 25 30 35 0.1 1 10 100 1000 Exchange time[min] dK58-2m cK58-2m wt-2m No. of protium atoms Fig. 7. Noncorrelated exchange kinetics of the folded conformations of b 2 -microglobulin (b2m) cleaved after Lys58 (cK58-b2m) (triangles), b2m with Lys58 deleted (dK58-b2m) (crosses), and wild-type (wt) b2m (circles). Shown are mass shifts (expressed as loss of protected protiated residues to adjust for differences in chain lengths) at 298 K as a function of time incubated in deuterated NaCl ⁄ P i . Stability of b 2 -microglobulin cleaved at Lys58 M. C. Mimmi et al. 2468 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS separations of samples kept at 298 K gave, for the slow-migrating conformer, concentrations of 38% ± 2% for cK58-b2m and 30% ± 4% for dK58-b2m (triplicate experiments ± SD), relative to the total peak area, independently of the total b2m concentra- tions used (examples are shown in Fig. 11A). CE sepa- rations are accomplished at low temperature in 10–12 min. Thus, solution states are sampled under dynamic conditions where the conformers are being separated from each other, whereas NMR spectra record steady-state solution distributions. Such differ- ences in experimental conditions may explain the differ- ences in the relative concentration estimates for the two conformers. However, both the NMR and CE approa- dK58-β2m compared to β2m-wt V9 Y10 S11 A15 N17 G18 G18 K19 S20 F22 L23 Y26 F30 D34 I35 E36 V37 D38 L40 K41 N42 G43 E44 R45 64I E47 L65 Y66 Y67 T68 E69 07F T71 P72 T73 D76 E77 Y78 A79 C80 R81 V82 N83 V85 T86 L87 P90 K91 V93 K94 W95 R97 D98 M99 -0,08 -0,06 -0,04 -0,02 0 0,02 0,04 0,06 0,08 0,1 0,12 residue ∆δΗα cK58-β2m compared to β2m-wt 1 I Q2 R3 T4 P5 K6 I7 Q8 V9 Y10 S11 R12 H13 P14 A15 E16 N17 G18 G18 K19 S20 12N 22F L23 N24 C25 Y26 V27 S28 G29 G29 F30 H31 P32 S33 D34 I35 E36 V37 D38 L39 L40 K41 N42 G43 G43 E44 R45 I46 E47 K48 V49 E50 H51 S52 D53 S55 F56 S57 D59 W60 S61 Y63 L64 L65 Y66 Y67 T68 E69 F70 T71 T73 E74 K75 D76 E77 Y78 A79 C80 R81 V82 N83 H84 V85 T86 87 L S88 Q89 P90 K91 I92 V93 K94 W95 D96 R97 D98 M99 -0,3 -0,25 -0,2 -0,15 -0,1 -0,05 0 0,05 0,1 0,15 0,2 residue ∆δΗα Fig. 8. Two-dimensional NMR study of b 2 -microglobulin (b2m) cleaved after Lys58 (cK58-b2m) and b2m with Lys58 deleted (dK58-b2m) at pH 7.4. The assigned backbone H a chemical shifts of 0.3 mM cK58-b2m at 298 K, and of 0.05 mM dK58-b2m at 310 K, are compared with the corresponding values of the wild-type (wt) species obtained at 310 K and pH 6.6. The DdH a values (p.p.m.) are reported as (Dvariant ) Dwt). Residue labels are omitted in regions where the resonance assignment was ambiguous. M. C. Mimmi et al. Stability of b 2 -microglobulin cleaved at Lys58 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2469 ches strongly support the notion of conformational het- erogeneity of the cK58-b2m and dK58-b2m variants. Conclusions Although cK58-b2m is unlikely to have a long lifetime in vivo, where the exposed Lys58 is rapidly cleaved off by endogeneous carboxypeptidase B activity [25], this variant was included in our study because it is more sta- ble in solution than dK58-b2m and thus more accessible to analysis. It has very similar characteristics in all the MS and CE analyses. However, we found for both b2m variants that the protein concentrations required for high-resolution NMR spectroscopy were detrimental to their stability in solution. The two cleaved b2m species have a pronounced propensity to undergo temperature- dependent unfolding and aggregation. In addition, the data show the occurrence of conformational heterogen- eity in cK58-b2m and dK58-b2m solutions, which is consistent with their thermal lability. Despite these diffi- culties, detailed characterization of the conformational states of the cK58-b2m and dK58-b2m variants has now been accomplished, and has made it possible, by reference to the NMR pattern of the DN3 variant of b2m, to identify a minor conformational species that also exists in the conformational equilibrium of the cleaved b2m variants. This conformer is a monomeric intermediate (I 2 ) occurring on the b2m-refolding path- way. These findings are consistent with the existence, in addition to the folded conformation, of a less abundant form with amyloidogenic features, which has also been suggested by CE experiments [9,10,22]. V37 Hg1* V37 Hg2* Y66 Hd* Y66 He* F70 Ha F70 Hd* F70 HN F70 Hz Y78 Ha Y78 Hd* Y78 HN A79 Ha A79 Hb* A79 HN W95 Hd1 W95 He1 W95 Hz2 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 H assignment ∆δH (p.p.m.) Fig. 9. Resonance doublings in b 2 -micro- globulin (b2m) cleaved after Lys58 (cK58-b2- m) indicating conformational heterogeneity. The proton chemical shifts of the alternative conformer (I 2 ) of cK58-b2m are compared with the corresponding values of the nat- ively folded form of cK58-b2m in the graph. The two conformers are recognized in TOCSY and NOESY maps, obtained at 298 K and pH 7.4. The DdH values (p.p.m.) are reported as (dI 2 ) dN), where N stands for the natively folded form. Only the most relevant deviations are shown. Resonance doubling observed elsewhere was less pronounced in terms of Dd. p.p.m. 10.410.610.811.0 p.p.m. 7.0 7.2 7.4 7.6 7.8 8.0 p.p.m. 10.410.610.811.0 p.p.m. 7.0 7.2 7.4 7.6 7.8 8.0 W95 N W95 I2 W95 N W95 N W95 N W95 I2 W95 I2 W95 I2 cK58 288K, pH 7.4 ∆N3 310K, pH 6.6 Fig. 10. Details of two-dimensional NMR NOESY maps of b 2 -microglobulin (b2m) cleaved after Lys58 (cK58-b2m) (left) and b2m devoid of the N-terminal tripeptide (DN3-b2m) (right), recorded at 500 and 800 MHz, respectively. The intraresidue connectivities H e1 –H d1 (top) and H e1 –H f2 (bottom) of Trp95 are indicated for the nat- ively folded form (N) and for the I 2 form, in equilibrium under the chosen experimental conditions. Stability of b 2 -microglobulin cleaved at Lys58 M. C. Mimmi et al. 2470 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... data clearly indicate that the overall folding pattern of the cleaved b2m variants is very similar to that of the wt protein In fact, distinct differences in the conformation of the variants are confined to the cleavage site region (the D–E loop) with additional involvement of the adjacent residues of strands D and E and the facing residues of loop B–C Accordingly, the noncorrelated amide hydrogen (1H... capillary temperature setting of 283 K The f and s conformers are indicated (B) Details of two-dimensional NMR TOCSY spectra of dK58-b2m and cK58-b2m obtained at 500 MHz, and b2m devoid of the N-terminal tripeptide (DN3-b2m) recorded at 800 MHz The specific intraresidue connectivities Hd*–Hz of Phe70 arising from the natively folded form (N) and I2 intermediate (I2) are labeled The unfolding processes that are. ..Stability of b2-microglobulin cleaved at Lys58 M C Mimmi et al Fig 11 Conformational heterogeneity of cleaved b2-microglobulin (b2m) by NMR and CE (A) CE analysis of b2-microglobulin cleaved after Lys58 (cK58-b2m) and b2m with Lys58 deleted (dK58-b2m) kept at 298 K and separated at 283 K Samples (2.5 mgÆmL)1) were injected for 2 s and analyzed at a constant current of 90 lA in 0.1 M phosphate, pH 7.4,... with temperature increase may be driven by the seeding probability, which increases with protein concentration and brings about increased recruitment of monomers or small oligomers onto the surface of soluble large aggregates The occurrence of large, well-defined aggregates has been recently demonstrated by size exclusion chromatography of dK58-b2m incubated at 310 K [10] The NMR data clearly indicate... marking b2m for clearance in the circulation and possibly failing in amyloidosis In any case, the results reported here provide a further basis for understanding the link between in vivo stability and the amyloidogenicity of conformationally unstable b2m variants Experimental procedures Protein purification b2m cleaved at Lys58 (cK58-b2m) and with an additional deletion of residue 58 (dK58-b2m) (Fig... 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2471 Stability of b2-microglobulin cleaved at Lys58 M C Mimmi et al the molecular destabilization characterized here and the formation of amyloid in vivo, these b2m molecular variants should be present in amyloid lesions from patients Conversely, given the propensity of b2m for specific cleavage, the pheomenon may be part of a physiological system... indicate that only slightly increased protection is conferred by the hydrogen bonds in wt b2m The reduced thermostability of b2m cleaved at Lys58 occurs despite an overall native- like folding and stems from a single cleavage in a rather mobile and exposed loop region [15,21] This cleavage is known to be mediated by complement enzymes that may be activated during inflammation To substantiate a relationship... lyophilized and redissolved in D2O Isotopic exchange was initiated by dilution (1 : 50) of the protiated protein solution with deuterated buffer, resulting in a final protein concentration of 20 lgÆmL)1 Typically, 10 lL of wt b2m, cK58b2m or dK58-b2m (c 1 mgÆmL)1 in protiated NaCl ⁄ Pi) was added to 490 lL of deuterated NaCl ⁄ Pi, pH 7.3 (value uncorrected for isotope effects) The proteins were incubated pairwise... conditions (pH 2.2 and 0 °C), where the exchange kinetics of main chain amide hydrogens is very slow Aliquots of fully deuterated wt b2m, cK58-b2m and dK58-b2m under quench conditions (pH 2.2 and 0 °C) were subjected to rapid desalting and they were observed to contain 88, 87 and 86 ±1 deuterium atoms, respectively Since wt b2m, cK58-b2m and dK58-b2m contain a total of 93, 92 and 91 main chain amide... was supported by MIUR (COFIN 2003) and by Sygesikringen ‘danmarks’ forskningsfond, Stability of b2-microglobulin cleaved at Lys58 Apotekerfonden af 1991, The Danish Medical Reseach Council, Lundbeckfonden, and M L Jørgensen og Gunnar Hansens Fond CarlsbergFondet is acknowledged for financial support to TJDJ The advice of Professor V Bellotti and the assistance of Dr A Makek are gratefully acknowledged . Variants of b 2 -microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous. values and thus confirm the retention of the main features of the native structure in both variants. The backbone H a chemical shifts of cK58-b2m and dK58-b2m