The Folding Pathway of the Antibody VL Domain

doi:10.1016/j.jmb.2009.07.075

J. Mol. Biol. (2009) 392, 1326–1338

Available online at www.sciencedirect.com

The Folding Pathway of the Antibody VL Domain Emma Rhiannon Simpson, Eva Maria Herold and Johannes Buchner⁎ Center for Integrated Protein Science Munich, Munich, Germany Department Chemie, Technische Universität München, D-85747 Garching, Germany Received 10 March 2009; received in revised form 22 June 2009; accepted 27 July 2009 Available online 6 August 2009

Antibodies are modular proteins consisting of domains that exhibit a βsandwich structure, the so-called immunoglobulin fold. Despite structural similarity, differences in folding and stability exist between different domains. In particular, the variable domain of the light chain VL is unusual as it is associated with misfolding diseases, including the pathologic assembly of the protein into fibrillar structures. Here, we have analysed the folding pathway of a VL domain with a view to determine features that may influence the relationship between productive folding and fibril formation. The VL domain from MAK33 (murine monoclonal antibody of the subtype κ/IgG1) has not previously been associated with fibrillisation but is shown here to be capable of forming fibrils. The folding pathway of this VL domain is complex, involving two intermediates in different pathways. An obligatory early molten globule-like intermediate with secondary structure but only loose tertiary interactions is inferred. The native state can then be formed directly from this intermediate in a phase that can be accelerated by the addition of prolyl isomerases. However, an alternative pathway involving a second, more native-like intermediate is also significantly populated. Thus, the protein can reach the native state via two distinct folding pathways. Comparisons to the folding pathways of other antibody domains reveal similarities in the folding pathways; however, in detail, the folding of the VL domain is striking, with two intermediates populated on different branches of the folding pathway, one of which could provide an entry point for molecules diverted into the amyloid pathway. © 2009 Elsevier Ltd. All rights reserved.

Edited by F. Schmid

Keywords: protein folding; protein stability; protein aggregation; fibril formation

Introduction The immunoglobulin (Ig) fold is a very widespread topology named after the domains contained within antibody molecules.1,2 This fold provides a source of topologically similar yet sequentially distinct proteins from which valuable information on differences in folding pathways can be gained. Antibody domains consist of a β-sandwich composed of two β-sheets. They share many structural features, including a disulfide bridge, which is buried in the hydrophobic core and often in close *Corresponding author. E-mail address: [email protected]. Abbreviations used: CDR, complementaritydetermining region; GdmCl, guanidinium chloride; PBS, phosphate-buffered saline; AFM, atomic force microscopy; PPIase, peptidyl prolyl cis–trans isomerase; β2m, β2-microglobulin; EDTA, ethylenediaminetetraacetic acid.

proximity to a conserved tryptophan residue, as well as the presence of several proline residues, one or more of which may be in cis conformation.3 These features make the folding of antibody domains more complex than the classical two-state folding observed for many small proteins.4 Proteins from the same family often fold via a similar pathway,5–7 and conservation of the folding nucleus in different members of the Ig superfamily has been shown.8–10 However, considerable differences in folding rates and the population of intermediates between family members can be observed, likely reflecting changes in the stability of different elements within the molecule that lead to different limiting factors.11–14 Detailed studies on the folding pathways of IgG constant domains illustrate the differences that closely related proteins can have in one fold. While they all show complex folding kinetics, with at least one phase rate being limited by proline isomerisation, the rate of nativestate formation and the number of pathways differ for the different domains. CL, the constant

0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.

1327

The Folding Pathway of the Antibody VL Domain

domain of the antibody light chain, folds within a few seconds via an intermediate, if all prolines are correctly isomerised.15,16 The folding of the CH2 domain (second constant domain of the antibody heavy chain) occurs on a similar timescale, also involving a population of intermediates,17 but the limiting factor for a subset of molecules here is cis–trans proline isomerisation, compared to the formation of a native-state cis-proline in the other domains. The CH3 domain (third constant domain of the antibody heavy chain) folds via at least two parallel pathways on a much slower timescale.18 In this case, prolyl isomerisation is closely linked to homodimerisation. The β-barrel structure of the variable domain of the light chain VL belongs to a subclass different from those of constant domains. It is made up of nine β-strands in a 4 + 5 orientation (ABED/CC′C″FG), while the constant domains are typified by seven strands in a 4 + 3 orientation (ABED/CFG).1 A further important difference between constant and variable domains is the presence of complementaritydetermining regions (CDRs) in the latter. These are loop structures connecting the β-strands, which lend antibodies their antigen-binding function. Engineering of these regions has allowed tailor-made binding specificities to be created.19–22 Previous studies on variable domains have suggested that their refolding kinetics reflect a complex folding pathway,23,24 yet a detailed characterisation is still missing. More recent studies have identified a cis-proline residue at position 95 as rate limiting for the folding of both an isolated VL domain and the VL fragment within a single chain Fv.25–28 The emergence of protein folding diseases involving this domain29,30 provided an impetus for further understanding of the factors contributing to or undermining the stability and solubility of the VL domain. Most prevalent is the fatal disease light chain amyloidosis, which results from overproduction of a monoclonal light chain that is prone to misfolding and leads to deposits of amyloid fibrils in organs such as the kidneys.31,32 Examination of these deposits has shown them to primarily contain the variable domain.29 Constant antibody domains appear to be much less susceptible to amyloid formation, with the CL domain featuring structural elements that seem to protect it from misfolding,33 suggesting that despite the similar topology, some factors within the VL domain can predispose it to this state. The fact that the majority of light chains are resistant to amyloid formation has allowed comparisons of amyloidogenic and nonamyloidogenic VL domains to attempt to identify the cause of this predisposition.34–39 It has been suggested that the formation of amyloid fibrils does not begin from the native state of a protein, but more likely from a partially folded state or an intermediate state.40–42 Mutations and changes in conditions that lead to destabilisation of the native state may therefore serve to increase the population of an intermediate state, thereby enabling more molecules to be diverted into an amyloid-forming pathway.43,44

This study presents a detailed characterisation of the folding pathway of a VL domain. We show that the fibrillar state is accessible for this domain. The folding pathway is relatively complex and shows features distinct from those of the CL, CH2, and CH3 domains, with two intermediates populated on different branches of the folding pathway.

Results Structural characterisation of VL The VL domain used in this study is obtained from MAK33 (murine monoclonal antibody of the subtype κ/IgG1), which is directed against human creatine kinase.45 The crystal structure of the Fab fragment of this antibody shows the VL domain to have a well-defined β-sheet structure stabilised by a buried disulfide bridge.3 The isolated VL domain has a far-UV circular dichroism (CD) spectrum with a minimum at 218 nm (Fig. 1a) typical of a β-sheet protein, indicating that the domain is able to form the β-barrel structure in isolation; however, the shape of the spectrum suggests a large contribution of random coil, which may be due to long flexible loops of CDRs. The near-UV CD spectrum shows that the tertiary structure of the protein is well formed with a minimum at ~275 nm, typical of antibody domains (Fig. 1b).46,47 The domain contains two tryptophan residues at positions 35 and 94 (numbering adapted from the Kabat Database48): the former is buried in close proximity to the disulfide, and the latter is found in a solvent-exposed position in CDR3. The presence of a buried tryptophan makes a sensitive probe for studying the conformation of the molecule by fluorescence, as shown by the spectra in Fig. 1c. A large increase in intensity, accompanied by a small redshift in the maximum wavelength (from 352 to 355 nm), occurs upon unfolding of the protein in guanidinium chloride (GdmCl). The small size of the redshift is attributed to the relatively high wavelength of the native-state emission, which is likely due to the contribution of the exposed W94 residue. Size-exclusion chromatography with fluorescence detection was used to assess the oligomeric state of the protein. VL domains are often found as dimers,49,50 although the monomeric state is thought to be necessary for fibrillation.44,51 For the MAK33 VL domain, a single peak was obtained at the same elution time over a 100-fold concentration range (from 1 to 100 μM; data not shown), indicating that no change in the oligomeric state occurred over this concentration range. Due to interactions with the column, the VL domain eluted later than expected, precluding a molecular weight analysis. Analytical ultracentrifugation experiments proved the monomeric state of the protein up to a concentration of 1 mM (data not shown).

1328

The Folding Pathway of the Antibody VL Domain

Fig. 1. Spectroscopic characterisation of the MAK33 VL domain. Far-UV CD spectra (a), near-UV CD spectra (b), and intrinsic tryptophan fluorescence spectra (c) of native (continuous line) and denatured (dotted line; 2.5 M GdmCl) VL in PBS buffer at 25 °C. (d) GdmCl-induced unfolding transition of the VL domain (10 μM) monitored by tryptophan fluorescence at 355 nm (filled symbols) and far-UV CD at 215 nm (open symbols) under equilibrium conditions. The reversibility of the unfolding process is shown by the overlay of unfolding (squares) and refolding (circles) experiments in both fluorescence and CD. Fluorescence experiments were also performed at a 10-fold lower concentration (filled triangles), indicating no influence of higher-order processes on equilibrium unfolding. The continuous and broken lines show the fit from each method to a two-state mechanism.

Thermodynamic stability of VL Equilibrium unfolding and refolding experiments were carried out to determine the conformational stability of the VL domain. Intrinsic tryptophan fluorescence at 355 nm was primarily used to monitor changes in conformation due to the large difference in intensity between the native state and the unfolded state (Fig. 1c). A cooperative sigmoidal transition was observed in both the refolding direction and the unfolding direction with identical midpoints (Fig. 1d), indicating the reversibility of the unfolding process in GdmCl. When unfolding was performed at a 10-fold lower concentration, no change in midpoint was observed, suggesting that second-order or higherorder processes do not influence the equilibrium unfolding process (Fig. 1d). Assuming a two-state transition, the data were fitted using linear extrapolation,52,53 which yielded a stability of −17.4± 2.4 kJ mol− 1 and an m-value of 16.0± 1.9 kJ mol− 1 M− 1. Thus, VL appears to be slightly more stable than the CL domain (14.6 kJ mol− 1) and the CH2 domain (15.5 kJ mol− 1) from MAK33,16,17 although the values are comparable when the error is taken into account. Interestingly, the stability of MAK33 VL is lower than

that reported for many other VL domains (approximately 25–35 kJ mol− 1),24,41,50,54,55 but is similar to that reported for amyloidogenic VL domains (approximately 15–20 kJ mol− 1).41,56 GdmCl-induced unfolding could also be monitored by far-UV CD, which yielded a reversible transition with a midpoint that is 0.2 M GdmCl higher than that observed using fluorescence (ΔGU = −19.8 ± 4.1 kJ mol− 1, m = 15.6 ± 3.0 kJ mol− 1 M− 1; Fig. 1d). This could suggest the presence of an equilibrium molten globule state;57–59 however, due to the low signal difference between native CD spectra and unfolded CD spectra (Fig. 1a), the reliability of this result is not certain (see Discussion). Thermal unfolding was monitored by the change in ellipticity at 215 nm (data not shown) and was found to be irreversible. The melting temperature of 50.6 ± 1.1 °C was comparable to that of the MAK33 CL domain (51 °C).16 Amyloid formation of VL Due to the stability of MAK33 VL being in a range commonly observed for amyloidogenic VL domains, the propensity of the protein to form fibrils was

1329

The Folding Pathway of the Antibody VL Domain

This indicates that the amyloid pathway is accessible for this protein, despite being not significantly populated at physiological pH. Together with the low stability, these features make this protein an excellent candidate for kinetics studies aiming to determine the folding pathway and to identify possible entry points into the amyloid pathway. Refolding and unfolding kinetics

Fig. 2. AFM images taken from a sample of VL that had been incubating at (a) pH 7.4 in PBS buffer and (b) pH 2 in 25 mM sodium phosphate/25 mM sodium acetate at 37 °C for 2 weeks. At pH 7.4, only background deposits can be seen; at pH 2, short fibrils can be observed.

ascertained. The protein was incubated with shaking in phosphate-buffered saline (PBS) at pH 7.4 and 25 °C for many weeks. At several intervals, the sample was monitored by atomic force microscopy (AFM), which showed no evidence of fibril formation (Fig. 2a). This was also the case when samples were incubated at 37 °C. Despite destabilising mutations, many VL domains only showed evidence of amyloid formation at low pH.60–62 Therefore, experiments were carried out at pH 2 to determine whether the amyloid state was accessible for this protein. After 2 weeks of incubation, amyloid fibrils were indeed observed (Fig. 2b). Inspection of the protein by mass spectrometry and SDS-PAGE indicated that the fibrils contain full-length proteins and that no significant hydrolysis had occurred despite the long incubation period (data not shown).

To investigate the folding pathway of VL, we determined the kinetics of refolding and unfolding at different GdmCl concentrations. The protein was first unfolded in 2.5 M GdmCl, which was sufficient to fully denature it (cf. Fig. 1d). Once equilibrium had been reached, the protein was diluted manually into a refolding buffer, and the change in fluorescence intensity at 355 nm was monitored. Refolding of VL is a slow process compared to the refolding of other antibody domains, 15–17,23 taking many hundreds of seconds. For final GdmCl concentrations below 300 mM, three exponential phases, with maximum rates of λ1 = 0.1 s− 1, λ2 = 0.018 s− 1, and λ3 = 0.005 s − 1 , were observed (Fig. 3a). These account for all the expected fluorescence changes upon refolding of the protein, as seen by a comparison of actual and expected intensity changes (Fig. 3b). To observe more clearly the fastest phase λ1, rapid mixing techniques were employed. This phase was only visible below 500 mM GdmCl, with a maximum of ∼20% of the total amplitude. The slower two phases had approximately equal amplitudes at low denaturant concentrations before the amplitude of λ3 decreased to zero at around 300 mM GdmCl. Above this (in manual mixing), λ2 is the only observable phase. The rates of this phase show the strongest dependency on the GdmCl concentration, while λ1 and λ3 show only a small dependency (Fig. 3a). The former suggests that λ2 is associated with a conformational change, as decreases in rate upon increases in denaturant concentration are generally coupled with the burial of a hydrophobic surface area.63 To kinetically analyse the unfolding of VL, the native protein was diluted into varying concentrations of GdmCl and monitored changes in fluorescence. Between 1 and 1.9 M GdmCl, singleexponential kinetics were observed (open circles in λ3 come from the interrupted refolding experiment below) (Fig. 3a). These also show a linear dependence on the GdmCl concentration and, together with the refolding λ2, form a V-shaped ‘chevron’ plot. Linear extrapolation of each of the arms of the chevron plot to 0 M GdmCl yields the rates of the processes occurring in the absence of denaturant, which in this case are 0.02 and 0.00001 s− 1 for refolding and unfolding, respectively. Upon rapid mixing, a faster unfolding phase, λ3, accounting for less than 10% of the total amplitude, was observed. The rate of this phase had a strong dependency on the GdmCl concentration, and extrapolation back to 0 M denaturant yielded a rate constant of 0.0004 s− 1. Refolding and unfolding experiments were also monitored by far-UV CD in order to see whether

1330

The Folding Pathway of the Antibody VL Domain

suggests a significant accumulation of secondary structure early within the folding process. Contribution of proline isomerisation to the refolding process

Fig. 3. (a) Chevron plot showing the variation in rates obtained from exponential fits of refolding and unfolding kinetics with GdmCl concentration. The VL domain in 0 or 2.5 M GdmCl was diluted 100-fold into an unfolding or refolding buffer with varying GdmCl concentrations, respectively, and the change in fluorescence intensity at 355 nm was monitored. λ1 refolding (open triangles) and λ3 unfolding (half-closed circles) were obtained using stopped-flow rapid mixing, while λ2 (closed squares) and λ3 refolding (half-closed circles) were obtained using manual mixing methods. The open circles were obtained from interrupted refolding experiments. Fit lines were obtained from a global fit of λ2 and λ3 to a triangular mechanism. (b) Variation in initial (circles) and final (squares) fluorescence intensities derived from exponential fits to the refolding data. The initial intensities at low GdmCl concentration correlate well with the expected extrapolation of the final intensity of the unfolded state, implying that all expected fluorescence changes are observed. However, the extrapolation of CD data (triangles) suggests a significant accumulation of structure occurring within the dead time of mixing.

differences could be observed when secondary structural changes were monitored (data not shown). Only a single exponential, corresponding to λ2, was observed in both refolding and unfolding. The lack of other observable phases may be partially due to poor resolution resulting from a low signal change between the native state and the denatured state (Fig. 1a). However, inspection of the expected initial amplitude of the CD data based upon extrapolation from the unfolded state reveals that a significant amount of the expected signal change occurs within the dead time of mixing (Fig. 3b). This

Taken together, the observation of multiple phases in both refolding and unfolding kinetics suggests that the folding pathway is more complicated than a simple two-state process. A contribution of oligomerisation/aggregation reactions during refolding was ruled out by performing experiments over a 10fold concentration range (final protein concentration, 0.5–5 μM). Here, no differences in observed rate constants were detected (data not shown). One common source of slow refolding phases that has been seen to be rate limiting in other antibody domains is the cis–trans isomerisation of peptidyl– prolyl peptide bonds.15–18,23,27,64 The presence of a cis-proline in the native state suggests that isomerisation is likely to occur during the folding process due to the predominance of the trans conformation in the unfolded state. In the MAK33 VL domain, there are five proline residues, two of which are likely to be in cis conformation in the native structure (Pro8 and Pro95). These are highly conserved among both λ and κ light chains.27,48 In order to ascertain whether proline isomerisation was responsible for either of the slow phases observed in the refolding kinetics, we carried out experiments in the presence of the peptidyl prolyl cis–trans isomerase (PPIase) cyclophilin B.65 The PPIase had the effect of accelerating λ2, while λ3 was relatively unaffected (Fig. 4a). This was surprising since λ3 has only a slight dependence on GdmCl concentration (a marker for isomerisation reactions) and is the slower phase. Similar effects were also observed in the presence of a completely different prolyl isomerase, Cpr6, which is a yeast cyclophilin.66 The confirmation of acceleration being due to prolyl bond isomerisation was provided by conducting of experiments additionally in the presence of the PPIase inhibitor cyclosporin A. Under these conditions, the rates returned to the values observed in the absence of the catalyst. From the dependency of λ2 on the denaturant concentration, this phase appears to be also associated with a significant surface burial. The acceleration by PPIases suggests that prolyl isomerisation and folding are likely to be coupled in this phase. Supporting this notion, the folding rates observed are within a range commonly observed for prolyl isomerisation.67,68 To determine whether other slow equilibration processes in the unfolded state were responsible for one of the phases observed in the refolding kinetics, we carried out double-jump experiments.69 Here, the native protein was unfolded for variable aging times using a high denaturant concentration (2.4 M GdmCl) to ensure that unfolding was faster than the potential isomerisation phases λ2 and λ3 before being transferred to low denaturant conditions (0.05 M GdmCl). In the second step, the refolding of the protein was directly monitored by fluorescence. At long aging times, the unfolded state will

1331

The Folding Pathway of the Antibody VL Domain

which can be used to describe the formation of the unfolded state (Fig. 4b). The formation of each of the populations of unfolded molecules that form the three refolding phases occurred with approximately the same rate (∼ 0.015 s− 1), corresponding most closely with λ2 (unfolding) from single-jump experiments. The additional presence of λ3 in one or more of these phases cannot be ruled out on the basis of manual mixing experiments alone, precluding an interpretation of these results in relation to the folding pathway. Nevertheless, together with the PPIase experiment, these results suggest that both λ2 and λ3 are indeed folding phases, despite the lack of dependency on GdmCl concentration in the latter. The acceleration of λ2 by PPIases suggests that proline isomerisation is involved in this folding process but is likely to be kinetically coupled with a conformational change. Population of intermediate states

Fig. 4. (a) Effects on refolding rate constants upon addition of cyclophilin B. Closed squares and half-open circles denote the refolding rates of λ2 and λ3, respectively, in 0.1 M GdmCl in the presence of increasing amounts of cyclophilin B. (b) Double-jump experiment of VL. The native protein was unfolded in 2.4 M GdmCl for various aging times before being refolded in 0.05 M GdmCl. All refolding phases were observed at all aging times. The increase in amplitude of each of the refolding phases corresponding to λ1 (closed squares), λ2 (half-closed circles), and λ3 (open triangles) is shown.

have time to equilibrate; therefore, all processes should be observable. However, at short aging times, only directly unfolded molecules will be present to refold. This should distinguish between phases occurring due to refolding and phases occurring due to slow equilibration processes in the unfolded state. This experiment has the advantage that it can also rule out other slow equilibration processes, such as nonprolyl peptide bond isomerisation, that have been seen to occur in some proteins.70 In the case of VL, all three refolding phases were observed at all times during the double-jump experiment (Fig. 4b). This suggests that neither of the two slower folding phases results from slow equilibration processes in the unfolded state because a delay in the appearance of an isomerisation phase would be expected. In addition, the rate of the refolding is independent of the denaturation time. The amplitudes of each phase increased with the aging time of unfolding in an exponential manner,

In order to elucidate the nature of the refolding phases observed in single-jump experiments, we carried out interrupted refolding experiments (Ntests).71 Here, a triple-jump experiment, where the protein was initially completely denatured, was performed. It was then refolded for a variable aging time in 0.1 M GdmCl, followed by transfer to unfolding conditions (1.7 M GdmCl), and the resulting fluorescence change was monitored. This experiment is able to monitor the formation of the native state and to discriminate between processes that directly form native molecules and processes that do not. Unfolding from intermediates may also be observed. In single-jump experiments under these conditions, the unfolding of the VL domain fitted to a single exponential (rate = 0.0035 s− 1). This result was replicated in the interrupted refolding experiment at long aging times, as expected. At short aging times, a second exponential with a faster unfolding rate of 0.074 s− 1 was observed. The amplitude of this phase initially increased with time before decreasing to zero above 300 s of aging time (Fig. 5). This is consistent with the formation and decay of an intermediate state, which is not observable in single-jump experiments. During short-term refolding, some molecules only refold as far as an intermediate state; therefore, upon transfer to high denaturant conditions, unfolding from this state is observable. From these data, it is possible to deduce information about the location of the intermediate along the folding pathway. As aging time increases, the amplitude of the major unfolding phase increases in an exponential manner, corresponding to the formation of native molecules (Fig. 5). Formation of an obligatory on-pathway intermediate would result in a lag in the formation of the native state. Due to the relatively slow formation of the intermediate, this lag would be clearly visible (Fig. 5, broken line). The absence of a long lag strongly suggests

1332

The Folding Pathway of the Antibody VL Domain Table 1. Microscopic rate constants at 0 M GdmCl derived from the global fitting of λ2 and λ3 in Fig. 3 and derived free-energy values k (s− 1) IC–IN IN–IC IN–N N–IN IC–N N–IC U–ICa U–N (eqm fluorescence) U–N (eqm CD)

Fig. 5. Interrupted refolding of MAK33 VL. The equilibrium unfolded protein was diluted 1:25 into 0.1 M GdmCl to initiate refolding. After various aging times, the solution was further diluted into 1.7 M GdmCl to interrupt refolding. The change in amplitude of the unfolding phases λ2 (closed squares) and λ3 (open circles) monitored by fluorescence is plotted against aging time. The continuous and dot/dash lines represent a global fit to a four-state model (as shown in Fig. 6), yielding rate constants of 0.15 s− 1 (U–IC), 0.011 s− 1 (IC–N), 0.002 s− 1 (IC–IN), and 0.011 s− 1 (IN–N). For comparison, the broken line shows the expected rate of formation of native molecules in a three-state on-pathway model. The inset shows the first 60 s of the formation of native molecules to illustrate the fast lag.

that this intermediate is not obligatory. This leads to the possibility of an off-pathway intermediate or multiple routes to the native state. Global fitting of interrupted refolding data in Fig. 5 yields a better fit of the intermediate data to a triangular mechanism involving one intermediate compared to a mechanism incorporating an off-pathway intermediate; however, a much better fit is obtained when four states are included in the mechanism. This corresponds well with data from single-jump experiments where three exponential phases are observed (thus suggesting that four states are populated72). In this scenario, the best fit to the data suggests that an initial intermediate, IC, is populated before partitioning into two parallel pathways occurs (Fig. 6). This is manifested as a short lag in the interrupted refolding data before the native state is populated (Fig. 5, inset). The rates obtained from this fit

0.01622 4.26e−4 0.00524 2.39e−5 0.01789 2.107e−5 0.086 ± 0.015

m (kJ mol

−1

M− 1) ΔG (kJ mol− 1)

− 7.97 2.87 − 1.54 − 12.37 − 3.10 3.12

−9.0 ± 0.9 −13.4 ± 0.9 −16.7 ± 0.5

16.0 ± 1.9

−3.1 ± 4.2 −17.4 ± 2.4

15.6 ± 3.0

−19.8 ± 4.1

a

The rate constant for U–IC transition is estimated from the chevron plot, and the stability is estimated from the difference between the equilibrium stability (measured by CD) and the change in stability between IC and N.

correlate well with the rates observed in singlejump experiments. The initial step from U to the first intermediate is in agreement with λ1, while λ2 and λ3 represent the two routes that possibly lead to the native state. However, due to the lack of data points within the lag, an additional direct route to the native state cannot be ruled out. The single-jump kinetic data were then fitted globally. The scatter in λ1 precluded a four-state global fit; nonetheless, a good fit to λ2 and λ3 was obtained using a triangular model (see Materials and Methods). Fitting of the data to an off-pathway model was not possible, further confirming the validity of the suggested mechanism in Fig. 6. The fit suggests that λ2 corresponds to the folding between the first intermediate IC and the native state, whereas λ3 corresponds to the folding and unfolding of the second intermediate state, labeled IN (Table 1). Confirmation that the unfolding arm of λ3 corresponds to the unfolding of the non-obligatory intermediate IN was provided by performing the interrupted refolding experiment at a fixed aging time. The fully unfolded protein was refolded for 100 s in 0.1 M GdmCl before being transferred to an unfolding buffer. This allowed the intermediate state to be maximally populated (Fig. 5); therefore, the unfolding of this intermediate could be observed. By unfolding between 1.5 and 1.8 M GdmCl, the denaturant dependence of this unfolding was observed. This yielded a series of points that corresponds well to λ3 (Fig. 3a, open circles).

Discussion Nature of intermediate states

Fig. 6. Proposed model for the folding of VL. The unfolded protein collapses to an initial intermediate state before partitioning into two parallel pathways of folding: one directly to the native state and the other via a second intermediate.

Our data suggest that the VL domain folds via two intermediate states—the first being an obligatory intermediate before the folding pathway splits into two parallel pathways, one of which involves the population of a second intermediate state. From the data presented, it is possible to draw some inferences about the nature of each of the intermediate states.

The Folding Pathway of the Antibody VL Domain

The lack of GdmCl dependency of the rate of formation of the first intermediate IC suggests that a significant hydrophobic surface area is not buried in this step, consistent with a loosely defined structure with few tertiary interactions. This may result from the collapse of a polypeptide chain into a more compact state such as a molten globule-like state.59,73 This leads to the speculation that IC could correspond to the potential equilibrium intermediate that has been suggested to be populated from equilibrium unfolding transitions. For VL, the tertiary structure may unfold prior to the secondary structure, leaving an intermediate characterised by secondary structure interactions but few tertiary interactions,57,58 as seen by the accumulation of secondary structure within the dead time of mixing of CD refolding experiments. Only a small difference between the two equilibrium transitions was observed, suggesting a maximum intermediate population of 20% in the transition region. The rate constants obtained from global fitting (Table 1) allow the relative stabilities of the different states to be calculated. The stability change from IC to N is 16.7 kJ mol− 1 (Table 1), similar to the equilibrium ΔGU–N value (17.4 kJ mol− 1) obtained from fluorescence experiments. This suggests that the majority of fluorescence changes only occur after the formation of IC (as fluorescence is a reporter on tertiary structure changes), consistent with the observations noted above. Nonetheless, since λ1 in fluorescencemonitored kinetics is attributed to the U–IC transition, some fluorescence changes must occur in this step, albeit with a low amplitude. A comparison of the CD equilibrium unfolding free-energy change ΔGU–N and the kinetics ΔGIC–N allows a small stability of 3.1 kJ mol− 1 to be estimated (Table 1). The large error in this value is attributed to uncertainty in the CD equilibrium unfolding value. Similarly to IC, folding of the second intermediate IN to the native state via λ3 also displays little dependency on the denaturant concentration. This suggests that the non-obligatory intermediate is much more native like, since little additional hydrophobic surface burial is required to achieve the native state. An increase in stability of 13.4 kJ mol− 1 occurs upon the formation of the native state from IN, suggesting that, despite being a compact intermediate, significant stabilising interactions are yet to be formed. An unusually large negative mvalue for the unfolding of the native state to IN is also observed. This would suggest that significant burial of hydrophobic surface area occurs during this transition. While it is possible that IN represents an overcompacted intermediate state, it is likely that there is a significant error in this value due to insufficient data points. Comparison to other Ig-like domains The folding of the MAK33 VL domain has been shown to be complex, involving multiple pathways and a population of intermediate states. This is comparable to previous studies on other antibody

1333 domains where multiple folding phases have been observed.15–18 One important difference in the folding of VL is the lack of a distinct proline isomerisation limited phase. Data presented here have shown that proline isomerisation plays a role in the folding of VL; however, due to the overall slow folding of the protein, structure formation occurs on a similar timescale. This is in contrast to the CL and CH2 domains from MAK33, which are able to fold within a few seconds in the absence of incorrectly isomerised prolines. Despite differences in timescale, the pathways of folding of CL, CH2, and CH3 show similarities to that of VL in the population of multiple pathways. In all cases, a fast route to the native state is observed, with a second route observed to involve one or more intermediates. The slower route in the refolding of all the constant domains is limited by proline isomerisation. This differs from the folding of VL, where proline isomerisation appears to be coupled with the faster folding phase, corresponding to the formation of the native state directly from the collapsed intermediate IC. The intermediate IN, populated on the alternative folding pathway, appears to be independent of proline isomerisation; therefore, limitation in the folding rate must arise from a different conformational change. Recent studies comparing the amyloidogenic β2microglobulin (β2m) with the antibody CL domain have shown that two small helices at the base of the latter play an important role in guiding the folding of CL.33 The intermediate formed in CL already has these helices present, while β2m, which has the Ig fold, lacks these helices in its native structure. The intermediate populated on the β2m folding pathway appears to be a precursor to amyloid fibril formation,74,75 while CL is resistant.33 The VL domain also lacks these helices, which could be an important determinant in the ability of Ig folds to achieve the fibrillar state. In addition, the intermediate formed in the folding of β2m is native-like, similar to the second intermediate on the folding of VL. The differences in the folding of VL to other antibody domains, particularly in the nature of the intermediates, might underlie the reasons that VL is associated with fibril formation and other IgG domains are not. VL from MAK33 also exhibits differences in its folding pathway compared to other VL domains. Apparent differences may be due to the nature of CDR loops that are, by necessity, different in all VL domains. A systematic study of the contribution of different CDR loops has not been carried out; however, significant differences in the stability of isolated VL domains have been observed with different length loops,76 and replacement of the loops in one VL domain has significantly increased stability.77 Loop regions can be important in the initial stages of protein folding, bringing different regions of the protein into close proximity.78 The structural features or lack thereof in different CDR loop sequences may play a role in the formation of intermediates, providing a kinetic trap from which the protein must escape before being able to form the native state.

1334 The population of two intermediates in the refolding of the VL domain is interesting in relation to the propensity of this protein to form amyloid fibrils. Current understanding suggests that diversion into a fibril-forming pathway is likely to begin from an intermediate state rather than from a fully unfolded or native state.40–42 The population of two intermediates here gives two potential opportunities for the protein to be diverted. Whether either of these intermediates is significant in opening up a channel to an alternative pathway remains to be seen; however, it seems more likely that the first intermediate IC would be a candidate. The likely structure content is quite low and mainly made up of secondary structure interactions. This suggests a state that could require minimal rearrangement to form a prefibrillar conformation, particularly since the sequence of the protein is already predisposed to forming β-strands and turns. Previous equilibrium studies have identified similar intermediates upon either acid or GdmCl unfolding of amyloidogenic VL domains. 61,62,79 In the case of the recombinant amyloidogenic protein SMA, an unfolded but compact intermediate involving mainly secondary structure interactions was observed at pH 2.61 The intermediate detected under equilibrium conditions for the MAK33 VL domain appears to be similar to the SMA intermediate, despite differences in reported stabilities (which are likely to be due to differences between the different VL domains). The intermediate populated at pH 2 of the amyloidogenic VL domain from the mouse monoclonal antibody F11 also appears to be similar to the collapsed state observed for MAK33 VL,62 supporting the theory that this could be the amyloid precursor. Further studies will be required to confirm this. In summary, a detailed description of the folding pathway of an amyloid-competent VL domain has been presented here, showing a complex folding pathway and, in contrast to other antibody domains, a population of two intermediates. One appears to be a collapsed molten globule-like state, while the other is populated on a non-obligatory pathway and is more native like. These intermediates provide possible entry points into the alternative pathway that VL populates on the way to the formation of amyloid fibrils.

The Folding Pathway of the Antibody VL Domain

between the NcoI and HindIII restriction sites. The plasmid was transformed into Escherichia coli BL21 cells for expression at 37 °C. At an OD600 of 0.6–0.8, expression was induced using 1 mM IPTG. Cells were harvested after overnight growth, and inclusion bodies were prepared as previously described.16,17 The pellet was solubilised and unfolded in 25 mM Tris–HCl (pH 8), 5 mM ethylenediaminetetraacetic acid (EDTA), 8 M urea, and 2 mM βmercaptoethanol at 4 °C for 2 h. The soluble fraction was then injected onto a Q-Sepharose column equilibrated in 25 mM Tris–HCl (pH 8), 5 mM EDTA, and 5 M urea. The protein was eluted in the flow-through and diluted five times before being refolded by dialysis into 250 mM Tris–HCl (pH 8.0), 5 mM EDTA, and 1 mM oxidised glutathione. To remove misfolded aggregates and remaining impurities, the protein was cleaned using a Superdex 75 gel-filtration column (GE Healthcare, Uppsala, Sweden) equilibrated in PBS buffer. The recovery of intact protein was verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CD measurements CD measurements were carried out using a Jasco J-720 spectropolarimeter (Jasco, Grossumstadt, Germany) equipped with a Peltier element. Far-UV CD spectra were measured using 10 μM protein in a 1-mm pathlength cuvette between 260 and 190 nm. Near-UV CD was measured between 320 and 250 nm using 50 μM protein in a 5-mm cuvette. All spectra were accumulated 16 times and buffer corrected. Samples for equilibrium stability measurements were prepared as described below, measuring the ellipticity at 215 nm for 50 s. Thermal transitions were also recorded at 215 nm with a heating and cooling rate of 20 °C /h. Analytical gel filtration Measurements were carried out using a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) and an analytical Superdex 75 column (GE Healthcare) equilibrated in PBS buffer. A standard flow rate of 0.75 ml min− 1 was used. One-hundred-microliter samples of VL at concentrations of 1, 10, and 100 μM were injected into the column, and the elution profile was detected by fluorescence emission at 355 nm. Fluorescence measurements

Cyclophilin B and Cpr6 were kind gifts from Matthias Feige and Martin Hessling, respectively (Buchner laboratory). GdmCl (ultrapure) and cyclosporin A were purchased from Sigma (St. Louis, MO, USA). All other chemicals were purchased from Merck (Darmstadt, Germany). The concentrations of GdmCl solutions were determined from refractive indices.52 Unless otherwise stated, all measurements were carried out at 25 °C and pH 7.4 in PBS buffer.

Fluorescence measurements were carried out using a Spex Fluoromax I or II (ISA, Edison, USA), with excitation and emission slit widths of 3 and 5 nm, respectively. The protein concentration in a 1-cm quartz cuvette was 10 μM. The sample was excited at 280 nm, and spectra were recorded between 300 and 450 nm. Equilibrium unfolding and refolding transitions were carried out by denaturing 10 μM protein overnight at different concentrations of GdmCl (between 0 and 3 M GdmCl). Fluorescence intensity at 355 nm was measured for 50 s, and the average was taken. Analysis of data was carried out using the linear extrapolation method described previously.52,53

Cloning, expression, and purification of the VL domain

Fibril formation

The VL domain was amplified by PCR from the cDNA of murine MAK33 and cloned into a pET28a vector

Samples of VL at a concentration of 50 μM in PBS buffer (pH 7.4) or 25 mM sodium phosphate/25 mM sodium

Materials and Methods

1335

The Folding Pathway of the Antibody VL Domain

acetate (pH 2) containing 0.05% sodium azide were incubated with shaking at 500 rpm at either 25 or 37 °C for many weeks. At various intervals, 20 μl was removed and added to a mica disc for AFM measurements. The disc was washed three times with water and allowed to dry. AFM measurements were carried out with a Digital Instruments multimode scanning probe microscope (Veeco, Santa Barbara, CA, USA) in contact mode using DNP-S20 tips.

was performed using Berkeley Madonna†. The chemical reactions module was used to fit the former, assuming irreversibility of the folding reactions to reduce the number of parameters. The analytical solution to a threestate triangular mechanism, as previously described,72 was used for single-jump kinetic data. Briefly, λ1 and λ2 are the two solutions to the following quadratic equation:

Refolding and unfolding kinetics

where γ1 = kNIkIU + kNUkIU + kINkNU, γ2 = kNUkUI + kUIkNI + kUNkNI, and γ3 = kUIkIN + kIUkUN + kUNkIN. The solutions are additionally constrained due to the principle of microscopic reversibility within the triangle:

For refolding experiments, either 25 or 100 μM protein was denatured in 2.5 M GdmCl overnight and then diluted into a refolding buffer in a stirred cuvette. For unfolding kinetics, native protein was diluted 25-fold or 100-fold into an unfolding buffer. The final protein concentration was 1 μM. The change in fluorescence intensity over time was monitored at the unfolded maximum of 355 nm, after excitation at 280 nm. In order to assess the effect of prolyl isomerases, either cyclophilin B or Crp6 were added to the refolding buffer to a final concentration of 10–200 nM. To test the inhibition of PPIase, we added a 2-fold excess of cyclosporin A. In all cases, at least three traces were averaged. For CD refolding or unfolding experiments, a 25-fold dilution of either native or unfolded (in 2.5 M GdmCl) protein was made to a final concentration of 10 μM. The change in ellipticity at 215 nm was monitored in a 1-mm cuvette. Stopped-flow measurements Rapid mixing experiments were carried out using an SX18-MV stopped-flow apparatus (Applied Photophysics, Leatherhead, UK) in single mixing mode with a mixing ratio of 1:25. Protein (100 μM) was diluted into various concentrations of refolding or unfolding buffer. Fluorescence was excited at 280 nm, and a cutoff filter at 320 nm was used. Five traces were averaged in each case. Double-jump experiments Native protein was diluted 10 times into 2.4 M GdmCl to initiate unfolding. After an aging time between 5 and 1000 s, the sample was further diluted 50 times into 0.05 M GdmCl, giving a final protein concentration of 1 μM. Fluorescence changes in the second step were monitored under the conditions described above. Interrupted refolding experiments The VL domain was fully unfolded overnight in 2.5 M GdmCl. The experiment was initiated by diluting the protein 25 times into 0.1 M GdmCl. After an aging time varying between 5 and 2100 s, the protein was further diluted into 1.7 M GdmCl to interrupt the refolding process. Fluorescence changes in this step were monitored under the conditions described above. The final protein concentration was 1 μM. Analysis of kinetic data Exponential changes in fluorescence were fitted using Origin (OriginLab, Northampton, MA, USA). Global fitting of interrupted refolding data and chevron plots

E2 −E4ðkUI + kIU + kUN + kNU + kNI + kIN Þ + ðg1 þg2 þg3 Þ ¼ 0

ðkUI + kIN + kNU Þ=ðkIU + kNI + kUN Þ = 1 The fitting software does not yield errors in the individual parameters obtained; however, an RMS value for the overall fit of 0.41 was obtained, indicating a good fit to the data. Errors in free-energy values were estimated from Origin fits, where possible.

Acknowledgements We thank Matthias J. Feige and Titus M. Franzmann for helpful discussions and Julia Esser for preliminary work on VL. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 749). Funding of Emma Rhiannon Simpson by the Alexander von Humboldt Foundation is gratefully acknowledged.

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The Folding Pathway of the Antibody VL Domain

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