Temperature Effects on Refolding and Aggregation of a Large

(3) King, J.; Haase-Pettingell, C.; Gordon, C.; Sather, S.; Mitraki, A. In Protein. Folding: in vivo .... sic and dibasic sodium phosphate were purcha...
0 downloads 0 Views 94KB Size
Download PDF
Anal. Chem. 1998, 70, 730-736

Temperature Effects on Refolding and Aggregation of a Large Multimeric Protein Using Capillary Zone Electrophoresis Pamela K. Jensen and Cheng S. Lee*

Department of Chemistry and Ames Laboratory, USDOE, Iowa State University, Ames, Iowa 50011 Jonathan A. King

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Capillary zone electrophoresis (CZE) equipped with laserinduced fluorescence detection (LIFD) allows rapid and sensitive identification of folding and aggregation intermediates of P22 tailspike endorhamnosidase using the intrinsic tryptophan fluorescence. Conversion of the protrimer into the native tailspike protein, which is the rate-limiting step, can be quenched at 0 °C and results in a loss of the protrimer in the refolding pathway. Results obtained from CZE-LIFD and polyacrylamide gel electrophoresis indicate the loss of folding intermediates through the adsorption of polypeptide chains onto the wall of the refolding vial. Early aggregation intermediates are studied using a temperature-sensitive folding tailspike mutant which shifts productive folding to the aggregation pathway at restrictive temperatures. By monitoring refolding intermediates during the temperature shift experiments, the early monomeric intermediate is identified as the thermolabile junctional intermediate between the productive and aggregation pathways. After passing the stage for accumulation of thermolabile intermediate, an increase in refolding temperature from 10 to 35 °C is carried out for achieving the optimal refolding kinetics and yields of tailspike endorhamnosidase. A critical problem both in the biotechnology industry and in biomedical research is the incorrect folding and association of newly synthesized polypeptide chains, resulting in formation of insoluble aggregates. It has been particularly, though not exclusively, associated with expression of cloned genes in heterologous hosts.1-3 Studies of refolding of denatured proteins in both in vitro and in vivo folding pathways indicate that aggregates are formed from partially folded intermediates and not from mature native or fully unfolded proteins.4 (1) Marston, F. A. O. Biochem. J. 1986, 240, 1. (2) DeBernardez-Clark, E.; Georgiou, G. In Protein Refolding; Georgiou, G, DeBernardez-Clark, E., Eds.; ACS Symposium Series 470; American Chemical Society: Washington, DC, 1991; Chapter 1. (3) King, J.; Haase-Pettingell, C.; Gordon, C.; Sather, S.; Mitraki, A. In Protein Folding: in vivo and in vitro; Cleland, J. L., Ed.; ACS Symposium Series 526; American Chemical Society: Washington, DC, 1993; Chapter 2. (4) Mitraki, A.; King, J. Bio/Technology 1989, 7, 690.

730 Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

Many proteins can fold in vitro from a completely denatured state,5,6 providing evidence that the primary structure of a polypeptide chain may contain all the information required for folding. However, the rules governing the mechanisms by which the polypeptide sequence dictates the three-dimensional conformation are not fully understood. The study of protein refolding is necessary to understand these underlying mechanisms, as well as for the prediction of protein structure. Most techniques for characterizing protein folding reactions depend on direct measurement of some average physical properties that are sensitive to changes in protein structure. Examples include changes in optical density and steady-state fluorescence intensity.7-11 Capillary zone electrophoresis (CZE) is an attractive alternative to experimental observation and provides access to a cross section of the population of molecular states within an equilibrium system during a protein refolding reaction. Additionally, separation in CZE is a function of intrinsic molecular properties including size and charge of macromolecules. On the basis of CZE separation, Rush et al.12 observed a conformational change in R-lactalbumin upon raising the column temperature. The initially sharp peak of the native protein became broad and somewhat asymmetric when higher temperatures were applied, indicating the presence of both the folded and a conformationally altered species. The temperature-dependent unfolding transitions of lysozyme at low pH and the unfolding process of human serum transferrin in urea have been studied by Hilser et al.13 and Kilar and Hjerten,14 respectively. CZE-UV detection was also utilized for the analysis of bovine trypsinogen, which underwent oxidation from a fully reduced molecule through a distribution of intermediate species until it reached the disulfide bond conformation corresponding to the native structure.15 (5) Creighton, T. E. Proteins, Structures and Molecular Properties; W. H. Freeman and Co.: New York, 1983. (6) Richards, F. M. Sci. Am. 1991, (Jan), 54. (7) Hagerman, P. J.; Baldwin, R. L. Biochemistry 1976, 15, 1462. (8) Labhart, A. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 7674. (9) Galat, A.; Yang, C. C.; Blout, E. R. Biochemistry 1985, 24, 5678. (10) Zuniga, E. H.; Nall, B. T. Biochemistry 1983, 22, 1430. (11) Kelley, R. F.; Wilson, J.; Bryant, C.; Stellwagen, E. Biochemistry 1986, 25, 728. (12) Rush, R. S.; Cohen, A. S.; Karger, B. L. Anal. Chem. 1991, 63, 1346. (13) Hilser, V. J.; Worosila, G. D.; Freire, E. Anal. Biochem. 1993, 208, 125. (14) Kilar, F.; Hjerten, S. J. Chromatogr. 1993, 638, 269. S0003-2700(97)00884-6 CCC: $15.00

© 1998 American Chemical Society Published on Web 02/15/1998

Figure 1. Schematic of tailspike refolding mechanism.

In our laboratory, CZE equipped with laser-induced fluorescence detection (LIFD) provided a fast, sensitive means of analyzing folding and aggregation intermediates of phage P22 tailspike endorhamnosidase using the intrinsic tryptophan fluorescence.16 The P22 tailspike endorhamnosidase is a homotrimer consisting of three 666-amino acid polypeptide chains. Each of the 72-kDa monomeric subunits within the trimer is mainly composed of a parallel β-helix structure.17 The native trimer is thermostable, with a Tm of 88 °C, and is resistant to sodium dodecyl sulfate (SDS) and protease.18,19 Upon initiation of refolding, tailspike polypeptides rapidly fold into structured monomeric intermediates with a high content of secondary structure (Figure 1). These monomeric species associate to form the triple-chain defined folding intermediates, the protrimers, that are susceptible to SDS and protease. Conversion of the protrimer into the native, SDS and protease-resistant tailspike protein is the rate-limiting step in the folding pathway.20,21 The folding and aggregation pathways of P22 tailspike endorhamnosidase were well characterized both in vivo18,22-24 and in vitro20,21,25,26 (Figure 1). It has been stated that refolding of tailspike could essentially be quenched by lowering the temperature to 0 °C,22,27 thus trapping the intermediates in a detergentsensitive state. However, little is known about how such low temperatures may affect the distribution of folding intermediates and the competition between productive folding and aggregation reactions. Furthermore, an early monomeric intermediate of tailspike protein is thermolabile and has an increased tendency to form aggregates at higher temperatures24,25,27 (Figure 1). This is due in part to the higher collisional frequencies and the changes in protein conformation.28 (15) Strege, M. A.; Lagu, A. L. J. Chromatogr., A 1993, 652, 179. (16) Jensen, P. K.; Fan, Z. H.; King, J.; Lee, C. S. J. Chromatogr., A 1997, 769, 315. (17) Steinbacher, S.; Seckler, R.; Miller, S.; Steipe, B.; Huber, R.; Reinemer, P. Science 1994, 265, 383. (18) Goldenberg, D.; King, J. J. Mol. Biol. 1981, 145, 633. (19) Sturtevant, J. M.; Yu, M.-H.; Haase-Pettingell, C.; King, J. J. Biol. Chem. 1989, 264, 10693. (20) Seckler, R.; Fuchs, A.; King, J.; Jaenicke, R. J. Biol. Chem. 1989, 264, 11750. (21) Fuchs, A.; Seiderer, C.; Seckler, R. Biochemistry 1991, 30, 6598. (22) Goldenberg, D.; King, J. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3403. (23) Goldenberg, D. P.; Smith, D. H.; King, J. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 7060. (24) Haase-Pettingell, C. A.; King, J. J. Biol. Chem. 1988, 263, 4977. (25) Mitraki, A.; Danner, M.; King, J.; Seckler, R. J. Biol. Chem. 1993, 268, 20071. (26) Speed, M. A.; Wang, D. I. C.; King, J. Protein Sci. 1995, 4, 900. (27) Goldenberg, D. P.; Berget, P. B.; King, J. J. Biol. Chem. 1982, 257, 7864.

In this study, CZE equipped with tryptophan fluorescence detection allows rapid identification of transient partially folded intermediates formed during tailspike refolding and aggregation at various folding temperatures. Additionally, a temperaturesensitive folding (tsf) tailspike mutant that acts to further destabilize an early folding intermediate at restrictive temperatures (at or above 39 °C) shifts productive folding to the aggregation pathway before association into protrimer.23 CZE monitoring of aggregation intermediates and pathways is demonstrated using a tsf mutant at restrictive temperatures. Optimization of refolding kinetics and yields measured by CZE is established on the basis of temperature shifts. In comparison to the results obtained from SDS and native polyacrylamide gel electrophoresis (PAGE), CZE provides an effective technique for investigating the effects of folding temperatures on the distribution and competition among misfolding (aggregation) and productive folding. EXPERIMENTAL SECTION CZE-LIFD. The CZE apparatus was constructed in the laboratory using a CZE 1000R high-voltage power supply (Spellman High-Voltage Electronics, Plainview, NY). Fused-silica capillaries of 50 µm i.d. × 150 µm o.d. were obtained from Polymicro Technologies (Phoenix, AZ). The capillaries were cut to a length of 50 cm with a separation distance of 27 cm between the injection point and the LIFD system. A negative electric potential was applied at the detector end of the capillary for electrokinetic injection and for electrophoretic separation while the injection end was grounded. CZE separations were carried out in 25 mM sodium phosphate-140 mM urea at pH 7.6. The CZE apparatus was enclosed in an interlock box for operator safety. A cooling jacket was utilized to maintain a constant temperature of 10 °C at the inlet buffer reservoir and around most of the separation capillary during refolding measurements. The LIFD system originally designed by Yeung and co-workers29 was employed for measuring tryptophan fluorescence of refolding intermediates separated by CZE. The 248-nm line of a KrF excimer laser (Potomac Photonics, Lanham, MD) was used as the excitation source, and fluorescence emission was collected at 340 nm using a photomultiplier tube (Hamamatsu, Bridgewater, NJ). CZE-LIFD equipped with cooling jacket was described in detail elsewhere.16 Protein Denaturation and Refolding. The P22 tailspike endorhamnosidase was unfolded at room temperature in a solution of 6.4 M urea and 25 mM sodium phosphate at pH 3 for 1.5 h. Refolding was initiated by rapidly diluting the denatured protein with 25 mM sodium phosphate buffer at pH 7, pre-equilibrated at the desired refolding temperature. Based on a 45-fold dilution, the final tailspike and urea concentrations in the refolding reaction were 33 µg/mL and 142 mM, respectively. The progress of the refolding reaction was monitored by subjecting an aliquot of refolding sample to CZE separation at various reaction times. Refolding samples were also analyzed by both SDS and native PAGE. For SDS-PAGE analysis, 20-µL aliquots of refolding sample were placed in vials containing 10 µL of SDS buffer (0.16 M Tris-HCl at pH 6.8, 6% SDS, 6% dithiothreitol (DTT), and 18% glycerol) and were kept at 4 °C until (28) Zettlmeissl, G.; Rudolph, R.; Jaenicke, R. Biochemistry 1979, 18, 5567. (29) Yeung, E. S.; Wang, P.; Li, W.; Giese, R. W. J. Chromatogr. 1992, 608, 73.

Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

731

electrophoresed. For native PAGE analysis, 20-µL aliquots of refolding sample were placed in vials containing 10 µL of native buffer (15 mM Tris, 0.11 M glycine, and 30% glycerol) at 0 °C and the mixtures were immediately electrophoresed. Slab Gel Electrophoresis. A Bio-Rad Mini-Protean II dual slab cell (Hercules, CA) was used to perform all SDS and native PAGE experiments. The separation and stacking gels were cast as described in the instruction manual. Acrylamide concentrations for the separating gels were 7.5% for SDS and 9% for native, while the stacking gels were 3% for SDS and 4.3% for native. SDS gels were run at 150 V, and electrophoresis stopped when the bromophenol blue dye front reached ∼1 cm from the gel bottom. Native PAGE was carried out at 200 V for 2-3 h at 4 °C. Silver staining of protein bands was performed as described elsewhere.30 Quantitation of the gel bands was achieved by scanning the dry gels and analyzing the intensities using the NIH Image software. Chemicals. The P22 tailspike endorhamnosidase and one of its tsf mutants, H304, were prepared and purified by standard procedures in King and Yu.31 Ultrapure urea (ICN, Aurora, OH) was used for all denaturation and refolding experiments. Monobasic and dibasic sodium phosphate were purchased from Fisher (Fair Lawn, NJ). DTT, glycine, glycerol, and SDS were obtained from Sigma (St. Louis, MO). All solutions were prepared using water purified by a NANOpure II system (Dubuque, IA) and further filtered with a 0.22-µm membrane (Costar, Cambridge, MA). RESULTS AND DISCUSSION Productive Refolding of Tailspike Endorhamnosidase at 10 °C. Tailspike refolding was initiated by rapidly diluting denatured protein with refolding buffer of 25 mM sodium phosphate (pH 7) at 10 °C. The final tailspike and urea concentrations in the refolding reaction were 33 µg/mL and 142 mM, respectively. The refolding reaction was monitored using CZELIFD by electrokinetically injecting an aliquot of sample at various times after initiation of refolding. The early-eluting peak corresponded to the formation of structured monomers which appeared rapidly at the beginning of reconstitution (Figure 2A) and gradually decreased as the refolding reaction proceeded. A second, later-eluting peak quickly emerged in the electropherogram (Figure 2B), and the fluorescence intensity of the peak continued to increase until reaching a maximum around the refolding time of 1-2 h (Figure 2C). The late-eluting peak was formed by association from the monomeric polypeptides and represented a precursor (the protrimer) of the native tailspike protein trimer.18,21 The conversion of the protrimer into the native, SDS-resistant tailspike protein is the rate-limiting step in the folding pathway20,21 and occurs very slowly at temperatures below 20 °C.18 After the first 3-4 h of refolding, an additional peak arose at a migration time between the structured monomer and the protrimer (Figure 2D) and the peak intensity continued to increase during the rest of the refolding reaction (Figure 2E-G). This peak was rationalized to be the mature trimer and was confirmed by spiking with a native tailspike standard (data not shown). Furthermore, the decrease in tryptophan fluorescence intensity of the protrimer was (30) Sather, S. K.; King, J. J. Biol. Chem. 1994, 269, 25268. (31) King, J.; Yu, M.-H. Methods Enzymol. 1986, 131, 250.

732 Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

Figure 2. Monitoring refolding of tailspike protein at 10 °C by CZELIFD. Refolding samples were taken at (A) 1 min, (B) 1 h, (C) 2 h, (D) 4 h, (E) 6 h, (F) 8 h, and (G) 24 h after the initiation of refolding. Electrophoresis buffer, 25 mM sodium phosphate and 142 mM urea at pH 7.6; capillary, 50 cm (27 cm to detector) × 50 µm i.d. × 150 µm o.d.; applied voltage, -6.5 kV and 10 s for refolding sample injection, -6.5 kV for electrophoresis; total tailspike protein concentration, 33 µg/mL; fluorescence detection at 340 nm.

accompanied by the accumulation of native tailspike. The observed refolding kinetics in the formation of monomeric and trimeric intermediates and native tailspike were in good agreement with those reported in the literature.20,21,25 Quenching of Tailspike Refolding at 0 °C. To better characterize the protrimer, the in vitro folding reaction was quenched by lowering the temperature rather than by denaturing all of the precursors with SDS. The refolding was carried out by diluting the denatured protein with refolding buffer equilibrated at 10 °C and was allowed to refold for 1 h. At this time, the distribution of folding intermediates would largely be at the protrimer stage with a residual amount of structured monomer and very little mature trimer yet formed. The refolding temperature was then reduced to 0 °C using a thermostated water bath. Thus, the concentrations of folding intermediates present before the temperature shift should remain essentially constant and no further production of native tailspike should occur.22,26 Tailspike endorhamnosidase was allowed to refold for 1 h at 10 °C (Figure 3A). Upon shifting the refolding temperature to 0 °C and continuing the refolding reaction for another 1 h, the amount of protrimer, instead of remaining constant, was noticeably reduced (Figure 3B). The tryptophan fluorescence intensity of the protrimer peak continued to decrease over a period of several hours at 0 °C (Figure 3C,D). At the same time, there was a slight

Figure 5. Monitoring refolding of tailspike protein during the temperature shift experiment using SDS-PAGE. Lanes: 1, denatured tailspike control; 2, native tailspike control; 3, 1 h after the initiation of refolding at 10 °C; 4-7, refolding samples taken from 1, 2, 3, and 6 h after the temperature shift from 10 to 0 °C.

Figure 3. Quenching of tailspike folding reaction by reducing the refolding temperature from 10 to 0 °C. Refolding reaction was monitored by CZE-LIFD. Refolding sample was taken at (A) 1 h after the initiation of refolding at 10 °C. The temperature was then shifted to 0 °C and the reaction continued for (B) 1, (C) 2, and (D) 4 h after the shift. All other conditions were the same as in Figure 2.

Figure 4. Monitoring refolding of tailspike protein during the temperature shift experiment using native PAGE. Lanes: 1, native tailspike control; 2, 1 h after the initiation of refolding at 10 °C; 3-8, refolding samples taken from 1, 2, 3, 4, 5, and 6 h after the temperature shift from 10 to 0 °C.

increase in the concentration of structured monomer as analyzed by CZE-LIFD. Conversion of the protrimer into the native, SDSresistant tailspike protein was quenched at 0 °C together with a loss of the protrimer in the refolding pathway. Both native and SDS-PAGE were also employed for analyzing the same refolding samples. The native PAGE (Figure 4) displayed three major bands corresponding to the protrimer, trimer, and monomer components of tailspike endorhamnosidase.22,32 Upon reducing the refolding temperature to 0 °C, the staining intensities of the protrimer and trimer bands continuously decreased for a total of 6 h refolding at 0 °C (lanes 3-8). By comparing the band intensities shown in lanes 2 and 8, the (32) Speed, M. A.; Morshead, T.; Wang, D. I. C.; King, J. Protein Sci. 1997, 6, 99.

temperature shift from 10 to 0 °C resulted in an approximate loss of 47% for protrimer and 52% for trimer. A minor protein band that migrated ahead of the trimer was attributed to a dimer species.26,33 The concentrations of both the monomer and dimer bands increased during tailspike refolding at 0 °C. By adding up the band intensities of the monomer, dimer, protrimer, and trimer in lanes 2 and 8, a ∼25% reduction in total protein concentration was measured during the temperature shift experiments. A very faint protein band corresponding to the native, SDSresistant tailspike was observed and remained relatively constant in SDS-PAGE (Figure 5). A refolding yield of ∼5% was estimated for the refolding sample taken from 6 h after the temperature shift from 10 to 0 °C (lane 7). It has been reported that an additional species whose mobility appeared to be that of native trimer in native PAGE was possibly an intermediate between the protrimer and the native trimer.33 This intermediate was still SDS-sensitive and would be denatured into monomer in SDS-PAGE. This would explain the higher trimer concentration measured in native PAGE (Figure 4) than those by CZE-LIFD (Figure 3) and SDSPAGE (Figure 5). Misfolding aggregates as well as those refolding species that have not yet matured into native trimers, including single-chain and protrimer intermediates, were denatured by the detergent and easily distinguished from the native tailspike. The monomer band on the SDS gel (Figure 5) appeared to decrease gradually (lanes 3-7), ending with a concentration that was 23% less than that present before the temperature shift (lane 3). This large decrease in polypeptide concentration analyzed by SDS-PAGE was in good agreement with the results of native PAGE and CZE-LIFD. The blockage in the conversion of the protrimer into the native tailspike at 0 °C shifted the folding equilibrium toward the monomeric intermediates and promoted the loss of polypeptide chains through the adsorption of monomeric species onto the wall of the refolding vial. In fact, the SDS buffer was added to the vial at the end of a refolding reaction to recover any adsorbed protein chains. An intense monomer band did appear on the SDS-PAGE gel (data not shown), indicating the adsorption of tailspike protein onto the vial. To rescue tailspike protrimer and shift the folding equilibrium toward the formation of native tailspike, the refolding temperature was increased from 0 to 10 °C after the refolding reaction was quenched at 0 °C for 2 h (Figure 6A-C). As shown in Figure (33) Robinson, A. S.; King, J. Nature Struct. Biol. 1997, 4, 450.

Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

733

Figure 6. Rescue of tailspike protrimer by increasing the refolding temperature from 0 to 10 °C. Refolding reaction was monitored by CZE-LIFD. Refolding sample was taken at (A) 1 h after the initiation of refolding at 10 °C. The temperature was then shifted to 0 °C and the reaction continued for (B) 1 and (C) 2 h after the shift. The temperature was increased back to 10 °C and the reaction proceeded for another (D) 1, (E) 3, (F) 5, and (G) 24 h. All other conditions were the same as in Figure 2.

6D-G, the productive folding pathway was resumed and the conversion of the protrimer into the native tailspike resulted in a refolding yield of ∼40%. This refolding yield was ∼10% less than that obtained from the refolding reaction at 10 °C for 24 h (Figure 2G), indicating an irreversible loss of structured monomer and protrimer during the temperature shift experiments. The refolding kinetics and yields measured by CZE-LIFD were in good agreement with those obtained via SDS and native PAGE (data not shown). Competition between Productive and Aggregation Pathways at High Temperatures. Refolding yield of tailspike endorhamnosidase was strongly dependent on the reaction temperature. High refolding temperatures promoted protein selfassociation and aggregation. To monitor the early and rapid folding events at high temperatures, the refolding reaction was initiated by diluting denatured protein with refolding buffer preequilibrated at 35 °C and the refolding sample was immediately injected into the capillary. The refolding sample was reinjected three times, each at intervals of 3 min, and continuously analyzed using CZE-LIFD (Figure 7A). Besides the peaks corresponding to the folding intermediates of structured monomer and protrimer, there was another distinct species migrating after the protrimer. The accumulation of native tailspike, together with the disappearance of protrimer, already occurred within 30 min after the initiation of refolding at 35 °C (Figure 7B), indicating faster reaction kinetics at high temperatures. The refolding yield obtained at 1 h after the initiation of refolding (Figure 7C) was ∼26% less than that of tailspike refolding at 10 °C. The low refolding yield was attributed to the fact that more polypeptide 734 Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

Figure 7. Monitoring refolding of tailspike protein at 35 °C by CZELIFD. Refolding samples were taken at (A) 1 min, 3 min, 6 min, and 9 min, (B) 30 min, and (C) 1 h after the initiation of refolding. All other conditions were the same as in Figure 2. The brackets in (A) represent the part of electropherogram removed for aligning the last sample injection in (A) with the electropherograms shown in (B) and (C). The migration order: M (monomer), T (native trimer), P (protrimer), and A (aggregate).

chains were shunted to the off-pathway aggregation versus the productive refolding pathway. Two partially resolved components were observed in the electropherogram with migration times past those of native tailspike and protrimer. These components might be the early intermediates in the aggregation pathway. To further investigate the identity of these two additional peaks, a refolding reaction was carried out using a tsf tailspike mutant, designated H304. On the basis of the results of native PAGE, this tailspike mutant acts to further destabilize an early folding intermediate at restrictive temperatures (at or above 39 °C) and shifts productive folding to the aggregation pathway before association into protrimer.23 By refolding H304 for 2 h at 39 °C, a very small amount of native tailspike was formed, barely distinguishable above the background (Figure 8A). At the same time, similar aggregate peaks as the ones shown in Figure 7 were observed in the electropherogram. To optimize both refolding kinetics and yields of tailspike endorhamnosidase, the refolding reaction was initiated at 10 °C for 12 min. The refolding temperature was then increased to 35 °C for the rest of the refolding reaction. After the temperature shift, the refolding sample was injected four times, each at intervals of 3 min, and continuously monitored using CZE-LIFD (Figure 9A). The distribution of folding components, including monomer, protrimer, and native tailspike changed rapidly during the first 30 min after the temperature shift (Figure 9A,B). The refolding yield at 1 h after the temperature shift (Figure 9C) was estimated at ∼75% and was the best yield over those obtained at constant

Figure 8. Monitoring refolding of tsf H304 tailspike mutant at 39 °C by CZE-LIFD. (A) 2 h and (B) H304 native tailspike control. All other conditions were the same as in Figure 2. Table 1. Refolding Yields of Tailspike Protein Measured by CZE-LIFD refolding temp

refolding yield (%)

constant 10 °C rescue from 0 °C constant 35 °C temp shift from 10 to 35 °C

50 40 24 75

10 (Figure 2G) and 35 °C (Figure 7C). Refolding yields of tailspike protein obtained at different refolding temperatures are summarized in Table 1. It is known that an early monomeric intermediate of tailspike endorhamnosidase is thermolabile and has an increased tendency to form aggregates at higher temperatures.24,25,27 Thus, the refolding reaction was initiated at 10 °C during the formation and accumulation of early thermolabile monomer species. Once the folding reaction has reached the stage for the production of association competent monomer, the folding temperature was increased to 35 °C for the enhancement of multimer formation including the protrimer and native tailspike. Rapid multimer formation in the productive folding pathway also reduced the amount of monomeric intermediates adsorbed onto the wall of the refolding vial and increased the overall refolding yield of native tailspike. CONCLUSION Rapid identification of transient partially folded intermediates formed during protein refolding and aggregation has been difficult, particularly with separation methods relying on solid matrixes. CZE in combination with the intrinsic tryptophan fluorescence detection provides a fast sensitive means for identifying folding and aggregation intermediates and for studying the effects of folding temperatures on the distribution and competition among misfolding (aggregation) and productive folding. In comparison

Figure 9. Optimization of refolding kinetics and yields by increasing the refolding temperature from 10 to 35 °C after the passage of thermolabile monomeric intermediate. Refolding reaction was initiated at 10 °C and monitored by CZE-LIFD. The temperature was then shifted to 35 °C at 12 min after the initiation of refolding. Refolding samples were taken at (A) 3 min, 6 min, 9 min, and 12 min, (B) 30 min, and (C) 1 h after the temperature shift. All other conditions were the same as in Figure 2. The brackets in (A) represent the part of electropherogram removed for aligning the last sample injection in (A) with the electropherograms shown in (B) and (C). The migration order: M (monomer), T (native trimer), P (protrimer), and A (aggregate).

to the results obtained from SDS and native PAGE, CZE is demonstrated as an effective technique for the analysis of tailspike refolding. However, the application of CZE-LIFD alone would be difficult to analyze unknown refolding systems. In general, more than one analytical technique is needed to collect all the necessary information for the study of a new refolding system. Tailspike refolding at low temperatures reduces the amount of aggregate formation at the expense of slow refolding kinetics. The refolding yields at low temperatures suffer from the adsorption of monomeric species onto the wall of the refolding vial, instead of the generation of aggregated polypeptide chains. For tailspike and many other proteins, the off-pathway aggregation reaction proceeds from an early thermolabile intermediate at the junction with the productive pathway. By monitoring refolding intermediates during the temperature shift experiments, the early monomeric intermediate is identified as the thermolabile junctional intermediate between the productive and aggregation pathways for tailspike endorhamnosidase. Optimal refolding kinetics and yields of tailspike protein are obtained by initiating the refolding reaction at low temperature and increasing the refolding temperature after passing the stage for formation and accumulation of the thermolabile intermediate. ACKNOWLEDGMENT Special thanks to Dr. Anne Robinson (Massachusetts Institute of Technology) for providing P22 tailspike endorhamnosidase and Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

735

for helpful discussions. This work was supported by the Biotechnology Process Engineering Center at Massachusetts Institute of Technology through the National Science Foundation’s Engineering Research Center Initiative under the Cooperative Agreement CDR-8803014. P.K.J. is a recipient of the Graduate Assistance in Areas of National Need (GAANN) Fellowship from the U.S.

736

Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

Department of Education. C.S.L. is a National Science Foundation Young Investigator (BCS-9258652). Received for review August 14, 1997. Accepted December 1, 1997. AC970884D