A kinetically trapped intermediate of FK506 binding protein forms in vitro: Chaperone machinery dominates protein folding in vivo

Protein Expression and Purification 51 (2007) 80–95 www.elsevier.com/locate/yprep

A kinetically trapped intermediate of FK506 binding protein forms in vitro: Chaperone machinery dominates protein folding in vivo Martin A. Wear, Alan Patterson 1, Malcolm D. Walkinshaw

*

Institute of Structural and Molecular Biology, Structural Biochemistry Group, The University of Edinburgh, Michael Swann Building, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Received 12 May 2006 Available online 28 June 2006

Abstract We have characterised the stability, binding and enzymatic properties of three human FK506 binding proteins (FKBP-12) differing only by the length and sequence of their N-terminus. One construct has a short hexa-his tag (6H-FKBP12); the second longer fusion protein (6HL-FKBP12) contains an additional thrombin protease cleavage site; the third has the long fusion tag removed and is essentially native FKBP-12 (cFKBP12). The proteins were purified both under native conditions and also using a refolding protocol. All three natively purified proteins have, within experimental error, the same peptidyl–prolyl isomerase (PPIase) activity (kcat/Km  1 · 106 M1 s1), and bind a natural inhibitor, rapamycin, with the same high affinity (Kd  6 nM). However, refolding of the protein containing the longer tag in vitro results in reduced PPIase activity (the kcat/Km was reduced from 1 · 106 M1 s1 to 0.81 · 106 M1 s1) and a 6-fold affinity loss for rapamycin. Addition of both the long and short N-terminal his-tags slows the refolding kinetics of FKBP-12. However, the shorter his-tagged fusion protein regains fully native activity (P95%) while the longer regains only 80–85% of native activity. Equilibrium urea denaturation titrations, isothermal titration calorimetry (ITC), analytical gel-filtration, and fluorescence binding data show that this loss of activity is not due to gross misfolding events, but is rather caused by the formation of a stable but subtly misfolded protein that has reduced peptidyl–prolyl isomerase (PPIase) activity and reduced affinity for rapamycin. The difference in behaviour between the in vitro refolded and native forms is due to the dominant role of the cellular chaperone/folding machinery.  2006 Elsevier Inc. All rights reserved. Keywords: Hexa-histidine tags; FK506 binding protein; Refolding; Peptidyl–prolyl isomerase; Rapamycin

The strong interaction between proteins containing short poly-histidine tags and immobilized metal-ions (Co2+, Ni2+, Cu2+, Zn2+) is one the most utilized chromatographic methodologies for the affinity purification of recombinant proteins (for reviews see [1–5]). One of the main advantages of this purification method is that histagged proteins (most commonly N-terminal hexa-histidine tags) are little affected in terms of their activity and specificity. The small size and highly charged/polar nature of the poly-histidine tag usually ensure that tagged-protein *

Corresponding author. E-mail address: [email protected] (M.D. Walkinshaw). 1 Present address: School of Biological and Environmental Sciences, University of Stirling, Stirling, Scotland, FK9 4LA, UK. 1046-5928/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.06.019

activity is rarely affected, but there are rare instances of this not being the case [6]. These effects may be overcome by switching the terminus to which the his-tag is attached [7]. Additionally, the purification can be performed under denaturing conditions (usually 8 M urea or 6 M guanidine hydrochloride), and then refolding the protein in vitro, either on the matrix or after elution from the metal-affinity resin. This approach often increases the efficiency of extraction from the cell and markedly reduces the presence of co-purifying contaminants [1]. However, it has the pre-requisite that the denatured protein refold spontaneously back to its native and active conformation without the aid of ‘‘folding machinery’’. This often practically limits the use of denaturing purification conditions followed by refolding for small (up to 15 kDa) single domains proteins; larger,

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more structurally complex multi-domain proteins are less likely to refold correctly without the extensive folding and chaperone machinery that exists in vivo [8]. Here we present results in which the biochemical activities of proteins being studied were significantly altered due to the presence of his-tags. This altered activity only manifests itself after the proteins were refolded in vitro following purification under denaturing conditions (8 M urea) on Ni2+–nitrilotriacetic acid (Ni2+–NTA)2 resin, and appears to dependent on the length, and likely, the sequence composition of the affinity-tag. We carried out these studies using human FK506 binding protein (FKBP-12), a small (12 kDa) ubiquitous cytosolic protein with peptidyl–prolyl isomerase (PPIase) activity [9] that binds the immunosuppressant drugs FK506 and rapamycin [10,11]. FKBP-12 is implicated in pivotal events in the intra-cellular signaling pathways that lead to immunosuppression [12,13]. It is also an important regulator of intracellular Ca2+ release mediated via an interaction with ryanodine receptors in skeletal and cardiac muscle [14–16]. In eukaryotes the rapamycin FKBP-12 complex binds and inhibits TOR, a highly conserved atypical protein kinase that controls proliferative growth [12,13]. There is considerable interest in FKBP-12 as a target for anti-cancer and cardiac hypertrophy drug therapies [13]. The structure of human FKBP-12, unbound and complexed with FK506 and rapamycin, has been solved to atomic resolution [17–20]. Human FKBP-12 contains no disulphide bridges, and in the native state all seven proline residues in the proteins 107 amino acids are in the trans conformation [17,18]. FKBP-12 can be readily denatured and rapidly refolded in a highly reversible two-state folding pathway [21–26] and its PPIase activity can facilitate auto-catalysis of its own refolding [22,27]. Human FKBP-12 is thus a good candidate for purification with metal-ion affinity as a recombinant his-tagged protein under denaturing conditions. We generated two constructs to express and purify human FKBP-12 as N-terminal hexa-histidine tagged fusion proteins from Escherichia coli. with a view to producing protein for a series of structure-function studies with libraries of small molecule inhibitors. One construct has a short non-cleavable hexa-his tag (6H-FKBP12), while a longer fusion protein (6HL-FKBP12) has an additional thrombin protease cleavage site between the his-tag and the N-terminus of FKBP-12. The final yield of pure protein was increased by between 15% and 20% for both forms of his-tagged FKBP-12 when performed under denaturing conditions, compared to when purification was carried out under native conditions. However, the biochemical activity of 6HL-FKBP12 is altered when the purified protein is refolding in vitro. Refolded 6HL-FKBP12 2

Abbreviations used: Ni2+–NTA, Ni 2+–nitrilotriacetic acid; FKBP-12, FK506 binding protein; PPIase, peptidyl–prolyl isomerase; PCR, polymerase chain reaction; sALPF-pNA, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide; pNA, p-ntitroaniline; BSA, bovine serum albumin; ITC, isothermal titration calorimetry.

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shows a 20% reduction in enzymatic activity and binds a natural tight-binding inhibitor, rapamycin, with a 6fold less affinity. Addition of a his-tag slows the refolding kinetics of both the long- and short-tagged fusion proteins, but the shorter one regains fully native activity while the longer does not. Removal of the his-tag with thrombin completely reversed these effects returning the biochemical activity of the refolded FKBP-12 back to those of natively purified FKBP-12. Our results suggest that the longer affinity-tag interferes with the in vitro refolding pathway resulting in a kinetically trapped, but stable misfolded form of the protein that has reduced PPIase activity and reduced affinity for rapamycin.

Materials and methods Materials All chemicals used were of the highest grade available commercially. Plasmid construction The plasmids for expression of recombinant human FK506-binding protein (FKBP-12) were created by polymerase chain reaction (PCR) using a whole tissue human lung cDNA library (Stratagene) as a template with 5 0 -CG CGGATCCATGGGAGTGCAGGTGGAAAC-3 0 as the forward primer and 5 0 -CCCAAGCTTTCATTCCAGTTT TAGAAGC-3 0 as the reverse primer for insertion into a pQE-30 vector (Qiagen), and 5 0 -GGAATTCCATATGGG AGTGCAGGTGGAAAC-3 0 as the forward primer and 5 0 -GCGCCTAGGTCATTCCAGTTTTAGAAGC-3 0 as the reverse primer for insertion into a pET-15b vector (Novagen). The resulting PCR products were verified by sequencing in both directions, using ABI PRISM BigDye v3 Terminator Cycle Sequencing Ready Reaction Kit and an ABI PRISM 310 Genetic analyzer (Applied Biosystems). For generation of the short, non-cleavable N-terminal hexa-histidine tagged (N-term-MRGSHHHHHHGSM-) FKBP-12 (6H-FKBP12) the PCR product was digested with BamHI and HindIII, and ligated into a pQE-30 vector similarly digested. For generation of the longer N-terminal hexa-histidine tagged (N-term-MGSSHHHHHHSSGLVP RGSHM-) FKBP-12 (6HL-FKBP12) the PCR product was digested with NdeI and BamHI and ligated into a pET-15b vector similarly digested. This construct contains a thrombin protease cleavage site (underlined). Correct insertion of the coding region was verified by restriction digest and by sequencing the entire coding region in both directions. Protein expression and purification ˚ KTA-FPLC (PharAll purification was performed on A macia) equipment at 4 C.

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Purification under native conditions Recombinant proteins were expressed and purified to homogeneity from BL21 Star or BL21 Star (DE3) E. coli (Invitrogen), 6H-FKBP12 and 6HL-FKBP12, respectively. Two times TY media containing carbenicillin (100 lg ml1) was grown shaking (260 rpm) at 37 C until the A600nm was 0.6. Over-expression of recombinant FKBP-12 was induced by addition of IPTG to 1 mM and growth for a further 4 h at 37 C. Cells were pelleted by centrifugation and resuspended at 10% (w/v) in ice cold PBS, pH 7.4; 1 mM NaN3; and 0.1% TX-100; 100 lM PMSF; 100 lM benzamidine; 5 mM b-mercaptoethanol; plus protease inhibitors. Lysis was performed by sonication, 6 · 30 s bursts on ice, with 30 s cooling in between. Cellular debris was removed by centrifugation at 50,000g for 1 h at 4 C. The supernatant was applied to 5 ml of prewashed (in PBS, pH 7.4; 1 mM NaN3; and 0.1% TX-100; 100 lM PMSF; 100 lM benzamidine; 5 mM b-mercaptoethanol) Ni2+–NTA agarose (Qiagen). The resin was washed extensively with PBS, pH 7.4; 1 mM NaN3; 100 lM PMSF; 100 lM benzamidine; 5 mM b-mercaptoethanol and bound proteins were eluted with a gradient of imidazole from 10 to 250 mM in the same buffer at 1 ml min1. Relevant fractions were pooled, concentrated to 1 ml and loaded onto an S200 HiPrep gel filtration column (Vt  120 ml; 2.6 · 60 cm) (Pharmacia) pre-equilibrated in 50 mM Hepes, pH 8.0; 100 mM NaCl; 10 mM DTT; 1 mM NaN3; 0.5 mM EDTA; and 0.1 mM PMSF, at 4 C. The column was run at 0.5 ml min1 and fractions analysed by SDS–PAGE (15% acrylamide). His-tagged FKBP-12 was P95% pure as judged by SDS–PAGE. Fractions containing monomeric FKBP-12 were pooled and concentrated to 500 lM and stored on ice. Purification under denaturing conditions Expression was performed as above. Cell pellets were resuspended at 10% (w/v) in PBS, pH 8.0; 8 M urea; 5 mM b-mercaptoethanol, and left gently stirring for 30 min at room temperature. Cellular debris was removed by centrifugation at 50,000g for 30 min at room temperature. The supernatant was applied to 5 ml of prewashed (in PBS, pH 8.0; 8 M urea; 5 mM b-mercaptoethanol) Ni2+–NTA agarose (Qiagen), and the proteins purified according to standard protocols. Refolding of denatured proteins The concentration of denatured protein was adjusted to 1 mg ml1 and refolding induced by a rapid 1:10 dilution into 50 mM Hepes, pH 8.0; 100 mM NaCl; 10 mM DTT; 1 mM NaN3; 0.5 mM EDTA; and 0.1 mM PMSF, at 4 C followed by dialysis over night against 10 L of the same buffer at 4 C. The protein samples were centrifuged at 50,000g for 1 h at 4 C, to remove any precipitated material, concentrated to 1 ml and loaded onto an S-200 HiPrep gel filtration column (Vt  120 ml; 2.6 · 60 cm) (Pharmacia) pre-equilibrated in 50 mM Hepes, pH 8.0;

100 mM NaCl; and 5 mM DTT; 1 mM NaN3; 0.5 mM EDTA; and 0.1 mM PMSF, at 4 C. The column was run at 0.5 ml min1 and fractions analysed by SDS–PAGE (15% acrylamide). His-tagged FKBP-12 was P95% pure as judged by SDS–PAGE. Fractions containing monomeric FKBP-12 were pooled and concentrated to 500 lM and stored on ice. Removal of his-tag from 6HL-FKBP12 The hexa-histidine tag was removed from 6HL-FKBP12 with thrombin (Promega) according to recommended protocols. Following cleavage, PMSF was added to 0.2 mM and free his-tag and uncleaved 6HL-FKBP12 removed by binding to Ni2+–NTA agarose in PBS, pH 7.4; 5 mM bmercaptoethanol; and 0.1 mM PMSF. The unbound cleaved protein (cFKBP12) was concentrated to 200 ll and loaded onto an Superdex-75 HR-10/30 gel filtration column (Vt  24 ml; 1.0 · 30 cm) (Pharmacia) pre-equilibrated in 50 mM Hepes, pH 8.0; 100 mM NaCl; and 5 mM DTT; 1 mM NaN3; 0.5 mM EDTA; and 0.1 mM PMSF, at 4 C. The column was run at 0.65 ml min1 and fractions analysed by SDS–PAGE (15% acrylamide). cFKBP12 was P95% pure as judged by SDS–PAGE. Peptidyl–prolyl isomerase (PPIase) assay This assay determines the rate of the cis to trans conversion of the peptidyl–prolyl amide bond in the peptide substrate N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (sALPF-pNA) (BACHEM). Selective enzymatic hydrolysis of sALtransPF-pNA by a-chymotrypsin [28] releases p-ntitroaniline (pNA), the accumulation of which is monitored by the absorbance at 400 nm. sALPF-pNA, dissolved in 470 mM LiCl in 2,2,2-trifluroethanol (TFE) at 200 mM, was diluted to 4 mM in LiCl/TFE immediately before use. Reactions were conducted at 4 C in 50 mM Hepes, pH 8.0; 100 mM NaCl; and 5 mM DTT, in a total reaction volume of 1 ml, essentially as described [29–31] with minor modifications. Between 1 and 200 nM (final concentration) of the appropriate purified FKBP-12 (in 50 mM Hepes, pH 8.0; 100 mM NaCl; and 5 mM DTT) was incubated for 30 min at 4 C in the absence or presence of the indicated concentration of rapamycin. a-Chymotrypsin (Worthington) was then added to 50 lM, the solution mixed and added to 25 ll of 4 mM sALPF-pNA (in TFE/LiCl). The reaction mixture was rapidly mixed and the A400nm recorded in a thermostated Jasco V550 spectrophotometer at 4 C for 120 s with a reading taken every 50 ms, with a dead time of  1–2 s. The final total concentration of sALPFpNA was 100 lM. The initial linear portion of the slopes (0–2 s) were converted to rates in lM s1 using the absorbance at 400 nm and the extinction coefficient e400 nm = 10,050 M1 cm1. Km and kcat values for respective FKBP12 proteins were determined using a range of substrate and enzyme concentrations. The initial reaction rates, V0 (in lM s1), were plotted against the concentration of sALcisPF-pNA and the data least squares fit to

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Eq. (1) using Kaleidagraph v3.6 software (Synergy Software, reading, PA): V 0 ¼ ðk cat  ½FKBPÞ  ½ Sub0 =ð½Sub0 =K m Þ þ ð½Sub0  k thermal Þ;

ð1Þ

where [FKBP] is the concentration of the appropriate FKBP-12 protein, [Sub]0 is the initial concentration of sALcisPF-pNA, kthermal is the first order rate constant for background uncatalysed thermal isomerisation (0.0019 s1) calculated from similar experiments conducted in the absence of FKBP-12, kcat is the turnover number and Km is the Michealis constant. Determination of the Ki values for rapamycin The apparent equilibrium dissociation constant, Kiapp, for rapamycin was determined by a least squares fit of Eq. (2) to plots of the initial reaction rate (background thermal isomerisation rate subtracted), V0 (in lM s1) versus the rapamycin concentration (in nM). V o ¼ ðV i =2  ½FKBP  fð½ FKBP  ½Inhib  K iapp Þ p 2 þ ðð½FKBP  ½ Inhib  K iapp Þ þ ð4  ½FKBP  K iapp ÞÞg;

mixture was left for 600 s to equilibrate before measurement of the fluorescence. Any change in the corrected fluorescence signal was assumed to be directly proportional to the concentration of FKBP-12: rapamycin complex. The observed fluorescence was buffer background subtracted, and corrected for dilution and inner filter effects by Eq. (4). F obs ¼ fff þ ðfb  ff Þ  ððK d þ ½ FKBP þ ½RapaÞ p  ððK d þ ½FKBP þ ½RapaÞ2  ð4  ½FKBP  ½RapaÞÞ =2  ½FKBPÞÞg  ðfobs =eð2:303e295nm L½RapaÞ Þ þ ðRapfluor  ½ RapaÞ;

ð4Þ

where Fobs is the observed fluorescence signal at 340 nm, e295 nm is the ligand the extinction co-efficient at 295 nm (30,500 M1 cm1 for rapamycin,), L is the path length (0.5 cm), [Rapa] is the concentration of rapamycin, Kd is the apparent dissociation equilibrium constant for the complex and Rapfluor is a linear correlation factor related to the concentration dependent auto-fluorescence of rapamycin (3.78 nM1).

ð2Þ

where Vi is the reaction rate at zero inhibitor concentration, [Inhib] is the concentration of added inhibitor, Kiapp is the apparent equilibrium dissociation constant of the inhibitor. Correction to account for competition with sALcisPF-pNA substrate was preformed using Eq. (3): K i ¼ K iapp =1 þ ð½Sub=K m Þ;

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ð3Þ

where [Sub] is the initial ALcisPF-pNA concentration (between 43 and 64 lM; mean = 51.2 lM) and Km is the Michealis constant of the substrate for the appropriate FKBP-12 protein. Intrinsic tryptophan fluorescence titration binding assay Human FKBP-12 possesses a single tryptophan residue (Trp59) that is located at the base of the ligand-binding site, in a highly hydrophobic environment [18]. Binding of rapamycin and all other ligands causes a concentration-dependent quench in the emission of Trp59. Fluorescence emission spectra were obtained on a PTI Quantmaster spectrofluorometer (Photon Technology International, Santa Clara, CA) at 25 C, in a 3 ml cuvette under constant gentle stirring. Trp59 was excited at 295 nm (2.5 nm band pass), and the fluorescence was measured at 340 nm (5 nm band pass). The fluorescence at 340 nm of between 220 and 700 nM of the corresponding FKBP-12 protein was measured (60 s with a reading every 50 ms) in the absence or presence of increasing amounts of rapamycin, in 50 mM Hepes, pH 8.0; 100 mM NaCl; and 5 mM DTT; 1 mM NaN3; 0.5 mM EDTA; 1% ethanol. Each point was obtained from sequential titration of rapamycin (300 lM stock in the above buffer), to the same sample of FKBP-12. Following each addition, the reaction

Isothermal titration calorimetry (ITC) experiments ITC experiments were performed using a VP-ITC machine (Microcal Inc. Northampton, MA). Proteins were dialysed exhaustively at 4 C into 50 mM Hepes, pH 8.0; 100 mM NaCl; 1 mM NaN3; 0.5 mM EDTA before use in the titration experiments. All solutions were degassed under vacuum for 6 min with gentle stirring immediately before use. Because of the low solubility of rapamycin, it was used in the sample cell (at a concentration between 1 and 2 lM) and titrated with the appropriate FKBP-12 protein the injection syringe. The enthalpy of binding (DH, kcal mol1) was determined by integration of the injection peaks and correction for heats of dilution were determined in similar experiments minus rapamycin. The resulting corrected binding isotherm was fitted by nonlinear least-squares analysis to a variety of binding models using MicroCal Origin software. Urea denaturation experiments FKBP-12 shows a single sharp transition from folded to unfolded states upon titration with the denaturant urea [21]. The intrinsic fluorescence of Trp59, highly enhanced (6-fold) upon unfolding [21–23,25,27], was measured (with excitation at 295 nm, 2.5 nm slit width) at 354 nm (5 nm slit width) to monitor the transition. A stock solution of urea (10 M; 50 mM Tris, pH 7.5; 10 mM DTT) was diluted to obtain a range of concentrations in 50 mM Tris, pH 7.5; 10 mM DTT. The appropriate FKBP-12 protein in 50 mM Tris, pH 7.5; 10 mM DTT was diluted 1:20 in the appropriate concentration of urea and incubated for 90 min at 25 C. The fluorescence at 354 nm was then

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measured. The free energy of unfolding of proteins, DGu–f, in the presence of a chemical denaturant is related to the concentration of that denaturant [32], as described by Eq. (5). H2 O DGuf ¼ DGuf  ðm  ½UreaÞ;

ð5Þ

where DGu–f is the free energy of unfolding at a particular urea concentration, DGu–fH2O is the free energy of unfolding in water, m is a constant that is proportional to the increase in degree of exposure of the protein on denaturation, and [Urea] is the concentration of urea. The measured fluorescence at 354 nm, F354nm, of the appropriate FKBP-12 was plotted versus the urea concentration and least squares fit to Eq. (6) using the Kaleidagraph program:

Miscellaneous

F 354nm ¼ fðIntN þ SlopeN  ½UreaÞ þ fðIntU þ SlopeU  ½UreaÞ  eðmð½Urea½Urea50% ÞÞ g=RT g=1 þ efmð½Urea½Urea50% Þg=RT ;

Recovery of PPIase activity Aliquots of the diluted refolding protein mixture were taken and incubated for 10 s in 50 mM Hepes, pH 8.0; 100 mM NaCl; and 5 mM DTT; and 50 lM a-chymotrypsin, at 4 C, to degrade unfolded and partially folded molecules. This mixture was then added to 25 ll of 4 mM sALPF-pNA and the rate of the PPIase assayed as above. The initial rate (V0 in lM s1) of each reaction was normalized to the initial rate of the same concentration of the appropriate natively purified form of FKBP-12 assayed in a similar manner. The final urea concentration (0.2 M) has negligible effect on the PPIase activity of FKBP-12 (data not shown).

ð6Þ

where F354nm is the measured fluorescence, IntN and SlopeN are the intercepts and IntU and SlopeU are the slopes of the fluorescence baselines at low [Urea] and high [Urea] concentrations, respectively, [Urea]50% is the concentration of urea at which the protein is 50% denatured, and m is the constant from Eq. (5). Refolding kinetics Analysis of the refolding kinetics of FKBP-12 was performed as described [23,27] with modifications. Unfolded proteins, at the appropriate concentration (in 50 mM Hepes, pH 8.0; 10 mM DTT; and 6 M urea), were diluted 1:30 to give final conditions of 50 mM Hepes, pH 8.0; 100 mM NaCl; 10 mM DTT; and 0.2 M urea. When FKBP-12 refolds the fluorescence of Trp59 is heavily quenched [21,25,27]. The folding of FKBP-12 occurs in three kinetic phases—a rapid initial phase (k1) followed by two slower phases (k2 and k3) [22,25,27]. The multiphasic nature of the refolding reaction results from the fact that there is a heterogeneous population of molecules in the unfolded state due to the isomerisation of the peptidyl–prolyl bonds in the seven proline residues present in the sequence of FKBP-12. The two slower phases (k2, k3) are limited by a proline isomerisation step [22,25,27]. The fast phase (k1), readily detectable in stop-flow experiments [22,25,27] is almost complete within the dead time (3–5 s) of the manual mixing experiments performed in this study. As a result, the fluorescence quench time course in refolding kinetics experiments were fit with a double exponential to get apparent rate constants for the slow-rate limiting isomerisation phases. At high protein concentrations, >5 lM, the refolding is very rapid and can be fit with a single exponential. This likely represents the fast direct folding reaction of the protein molecules where all seven peptidyl–prolyl bond conformations are trans [22,25,27].

Tris/tricine SDS–PAGE (16.8% acrylamide) was performed as described [33]. The molecular weights of cFKBP12, 6H-FKBP12, 6HL-FKBP12 and rapamycin are 12,231, 13,205, 14,183 and 914.02 Da, respectively. Protein concentration was determined by measurement of absorbance at 280 nm and calculated using the extinction coefficient 9970 M1 cm1, or by BCA protein assay (Pierce) with bovine serum albumin (BSA) as a standard, where proteins were selectively precipitated with deoxycholate and trichloroacetic acid to remove interfering agents as described [34], prior to BCA assay. Both methods gave values for all three proteins that differed by only ±4.3%. Results and discussion Expression and purification of FKBP-12 We generated two constructs to express human FKBP12 as recombinant N-terminal hexa-histidine tagged fusion proteins in E. coli. One construct has a short non-cleavable hexa-his tag (6H-FKBP12), while a longer fusion protein (6HL-FKBP12) contains an additional thrombin protease cleavage site (Fig. 1A). Both his-tagged proteins were effectively purified using a simple two-step purification protocol; a Ni2+–NTA affinity step followed by a ‘‘polishing’’ gel filtration step (Fig. 1B, Table 1). The final levels of purity for 6H-FKBP12 and 6HL-FKBP12 were essentially identical (P95%, Fig. 1B, Table 1) using either native or denaturing purification conditions. However, the final yields of both his-tagged proteins purified under denaturing conditions were increased, on average, by 17% over that obtained under native conditions (Table 1). FKBP-12 is one of the largest proteins to show a highly reversible two-state unfolding pathway [21,23–27]. It also exhibits auto-catalytic refolding activity and regains native levels of PPIase activity upon refolding after denaturation in urea [27]. Thus, purifying our his-tagged FKBP-12 constructs under denaturing conditions appeared the best way to maximize the amount of protein available for structural studies. To ensure the protein’s activities were unaltered by the denaturation and refolding process, we assayed the bio-

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Fig. 1. Final purity of FKBP-12 fusion proteins. (A) Schematic diagram showing sequence of the N-termini of the three FKBP-12 fusion proteins used in this study. The thrombin cleavage site in 6HL-FKBP12 is shown underlined. (B) Tris/tricine SDS–polyacrylamide gel (16.8% acrylamide) [33] of the final purified preparations of the respective FKBP-12 protein, purified under native conditions. Five micrograms total protein was loaded in each lane, and densitometric analysis of similar gels indicated that protein preparations were P95% pure. (C) The A280 monitored Superdex-75 HR-10/30 gel-filtration elution profiles of refolded cFKBP (black), refolded 6H-FKBP-12 (red) and refolded 6HL-FKBP12 (gray) are shown. All three refolded FKBP-12 proteins elute as a single monodisperse species with apparent molecular masses clustered around the 13.7 kDa standard (Ribonuclease-A). One hundred and eighty microliters of each FKBP12 protein, at 100 lM, was loaded and the column run at 0.6 ml min1 in 50 mM Hepes, pH 8.0; 100 mM NaCl; 5 mM DTT; 1 mM NaN3; and 0.5 mM EDTA, at 4 C. 0.5 ml fractions were collected. The elution volumes of molecular mass standards and the void volume (V0) are indicated. The inset shows a plot of Kav versus the log10 of the molecular mass of the indicated molecular mass standards. Red crosses indicate the elution positions of the three refolded FKBP-12 proteins in relation to the standards. Elution volumes and apparent molecular masses of cFKBP12, 6H-FKBP12 and 6HL-FKBP12 were 13.39 ml, 13.9 kDa, 13.35 ml, 14.3 kDa and 13.41 ml, 14 kDa, respectively. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

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Table 1 Purification of 6H-FKBP12 and 6HL-FKBP12, under native or denaturing conditions Fraction

Total protein (mg)a 6H-FKBP12/6HL-FKBP12

Purity (%)b 6H-FKBP12/6HL-FKBP12

Native conditions Supernatant Pooled Ni2+–NTA fractions Pooled S-200 fractions

338/342 42/40 36/37

9/8.8 87/90 P95/P95

Denaturing conditions Supernatant Pooled Ni2+–NTA fractions Pooled S-200 fractions

398/406 54/57 43/44

11/12 94/93 P95/P95

Fractionation was performed on cell pellets obtained from 2 L of E. coli culture. a Determined from BCA protein assay measurements. b Determined by densitometry of appropriate lanes on SDS–PAGE and Tris/tricine SDS–PAGE gels, similar to those shown in Fig. 1B.

chemical activities of 6H-FKBP12 and 6HL-FKBP12, as well a protein with the his-tag removed (cFKBP12), purified under native conditions (Fig. 1B) and compared them to the corresponding activities of proteins purified under denaturing conditions and then refolded in vitro. Before conducting any biochemical assays, we verified that the refolded protein preparations were monomeric and contained no other oligomeric/aggregate species. All three refolded FKBP-12 proteins behave as highly monodisperse species, eluting as single sharp peaks from a Superdex-75 HR-10/30 gel-filtration column, with the apparent molecular masses for all three proteins clustered around the 13.7 kDa standard (Ribonuclease-A) (Fig. 1C). These results suggest that all three refolded proteins are monomeric proteins and their apparent molecular masses agrees well with their theoretical molecular masses, 12.3, 13.2 and 14.2 kDa, cFKBP12, 6H-FKBP12 and 6HL-FKBP12, respectively (Fig. 1C). All forms of FKBP-12 purified under native conditions show essentially identical elution profiles to the refolded proteins (data not shown). Refolded 6HL-FKBP12 shows reduced PPIase activity The peptidyl–prolyl imide bond adopts both cis and trans conformations. The PPIase activity of FKBP-12 catalyzes the rotation about this imide bond [9], speeding up attainment of the cis–trans equilibrium. Measurement of PPIase activity exploits the high conformational selectivity of a-chymotrypsin, which cleaves the imide bond of a chromogenic prolyl peptide substrate only in the trans conformer [28], releasing, in this case, p-nitroaniline (pNA). The rate of accumulation of pNA is monitored by absorbance at 400 nm and directly correlates to the rate of PPIase activity [28,30]. Using this assay, we determined the Km and kcat kinetic constants for both the natively purified and the refolded forms of FKBP-12. The enzymatic activities of natively purified 6H-FKBP12, 6HL-FKBP12 and cFKBP12 are essentially indistinguishable from each other (Fig. 2A). The mean values for both the kcat and Km varied minimally around 300 s1 and 300 lM, respectively (Table

2], agreeing with the range of literature values for these kinetic rate constants for various eukaryotic FKBP-12 proteins (kcat; 300–600 s1 and Km; 200–600 lM) [30,35–37]. The catalytic efficiencies (kcat/Km) for all three proteins varied minimally around 0.95 · 106M1 s1 (Table 2), in agreement with the published value, 1.2 · 106M1 s1 [37], for human FKBP-12 and the same peptide substrate at the same temperature. The activities of refolded 6HFKBP12 and cFKBP12 were essentially unchanged, both compared to each other and to the corresponding protein purified under native conditions. The kcat and Km values varied minimally around 300 s1 and 300 lM, respectively, whilst the kcat/Km for both proteins varied minimally around 1.0 · 106M1 s 1 (Fig. 2B; Table 2). In contrast, however, the PPIase activity of refolded 6HL-FKBP12 was very obviously reduced (red line, Fig. 2B; Table 1). The turnover number (kcat) of 6HLFKBP12 was reduced markedly from 300 s1 to a mean value of 232 s1, while the Km was reduced very slightly from 300 lM to mean value of 282 lM (Table 1), giving a protein with an apparent 21% reduction in catalytic activity (the kcat/Km is reduced from 1 · 106 M1 s1 to 0.81 · 106 M1 s1; Table 2). A double reciprocal plot of the initial isomerisation rate versus the substrate concentration is shown in the inset to Fig. 2B. The enzymatic efficiency of 6HL-FKBP12s PPIase activity has been reduced, while the Km is essentially unchanged, suggesting that the apparent inhibition is not occurring via direct competition for substrate at the active site, but by some non-competitive manner. The addition of the longer his-tag reduces the effectiveness of FKBP-12’s PPIase activity, whereas the short one does not. However, this effect only manifests itself after refolding of the enzyme in vitro as natively purified 6HL-FKBP12 exhibited native activity. Removing the long his-tag alleviates this apparent loss of enzymatic activity as refolded cFKBP12 shows no difference in activity compared to its natively purified counterpart (Fig. 2B; Table 2). Similar experiments performed with different protein concentrations gave essentially identical results (data not shown).

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Fig. 2. Comparison of the biochemical activity of cFKBP12, 6H-FKBP12 and 6HL-FKBP12, purified under native conditions (A, C, and E) or denaturing conditions and then refolded (B, D, and F). A–F: filled circles, cFKBP12; open triangles, 6H-FKBP12; open circles, 6HL-FKBP12. (A and B) The initial velocity (V0, lM s1) of FKBP-12 (42 nM) catalysed cis–trans isomerisation of the tetrapeptide substrate sALPF-pNA is plotted versus increasing concentrations of the substrate. The solid lines are a best fit to Eq. (1) (see Materials and methods) giving the values for kcat and Km shown in Table 2. Inset in (B) shows a double reciprocal plot of the data for 6HL-FKBP12, suggesting a predominantly non-competitive element to the inhibition of refolded 6HL-FKBP12. (C and D) Inhibition of PPIase activity of FKBP-12 (42 nM) by rapamycin. The solid lines are a best fit to Eq. (2) (see Materials and methods). The values for the equilibrium dissociation constant for rapamycin inhibition (Ki) are shown in Table 2. (E and F) The intrinsic tryptophan fluorescence of FKBP-12 (0.56 lM) at 340 nm is plotted versus the rapamycin concentration. The solid lines are a best fit to Eq. (4) (see Materials and methods) giving the values for the equilibrium dissociation constant for rapamycin (Kd) shown in Table 2. All data points shown are the mean ± SE from three repeats. In (F) error bars are omitted for clarity.

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Table 2 Biochemical activity comparison of cFKBP12, 6H-FKBP12 and 6HL-FKBP12, purified under native conditions or refolded following urea denaturation Protein Native cFKBP12 6H-FKBP12 6HL-FKBP12 Refolded cFKBP12 6H-FKBP12 6HL-FKBP12

kcat (s1)

Km (lM)

kcat/Km · 106 (M1 s1)

Ki (nM)

Kd (nM)

310 ± 29 292 ± 42 289 ± 38

325 ± 27 301 ± 44 304 ± 34

0.95 ± 0.15 0.97 ± 0.24 0.95 ± 0.21

4.7 ± 1.2 5.1 ± 2.2 4.1 ± 2.0

7.4 ± 4.2 7.8 ± 3.7 6.2 ± 2.2

308 ± 36 310 ± 31 232 ± 37a

297 ± 37 303 ± 35 282 ± 22

1.03 ± 0.21 1.02 ± 0.19 0.81 ± 0.17

7.5 ± 1.9 6.1 ± 2.1 23 ± 3.7b

8.7 ± 3.5 6.8 ± 1.2 45 ± 7b

a

The kcat value for refolded 6HL-FKBP12, compared to the kcat values for refolded 6H-FKBP12 and cFKBP12, has a P value 60.15. The Ki and Kd values for refolded 6HL-FKBP12, compared to the Ki and Kd values for refolded 6H-FKBP12 and cFKBP12, have P values 60.05. All values are mean ± SE, n = 3. b

Refolded 6HL-FKBP12 binds rapamycin with reduced affinity We next assayed the ability of rapamycin to inhibit the PPIase activity of the various FKBP-12 constructs. Rapamycin is a lipophilic macrolide drug, produced by the bacterium Streptomyces hygroscopicus [38] that binds tightly to the catalytic site inhibiting FKBP-12 [10]. Fig. 2C and D shows the effect of incubating increasing concentrations of rapamycin on the respective FKBP-12 proteins ability to catalyse the cis–trans isomerisation of the peptidyl–prolyl bond in the tetra-peptide substrate. All three natively purified proteins showed essentially identical inhibition curves (Fig. 2C) and the values determined for the equilibrium dissociation constants for inhibition (Ki) for rapamaycin were all in the 4–6 nM range (Table 2). Similar values determined by this assay are reported in the literature for human FKBP-12 [35,36,39,40]. Refolded 6HFKBP12 and cFKBP12 were inhibited by rapamycin in a manner indistinguishable not only from each other but also their respective natively purified counterparts (Fig. 2D; Table 2). However, refolded 6HL-FKBP12 shows a significantly altered rapamycin-inhibited PPIase profile (red line, Fig. 2D). The initial rate of the PPIase activity is much reduced, for the same total concentration of enzyme, and relatively more rapamycin is required to inhibit 6HLFKBP12 to the same extent as either of the other two refolded proteins. The mean Ki value for refolded 6HLFKBP12 binding to rapamycin is 23 ± 3.7 nM; 4-fold weaker compared to the other FKBP-12 proteins, purified either under native or denaturing conditions (Table 2). This loss of affinity for rapamycin is only apparent upon refolding of 6HL-FKBP12. It again appears that the loss of function upon refolding is a property of the long his-tag as refolded 6H-FKBP12 binds rapamyicn with native affinity (mean Ki = 6.1 ± 2.1 nM; Table 2), whilst removal of the tag allows refolded cFKBP12 to bind rapamycin with affinity indistinguishable from its natively purified counterpart (mean Ki = 7.5 ± 1.9 nM; Table 2). Similar experiments performed with different protein concentrations gave essentially identical results (data not shown).

Next we assayed the physical interaction of rapamycin with both natively purified, or denatured and refolded FKBP-12 forms, by intrinsic tryptophan fluorescence (Fig. 2E and F). Human FKBP-12 possesses a single tryptophan residue (Trp59) located at the base of the ligandbinding/active site in a highly hydrophobic environment [18]. The binding of rapamycin (and all other ligands) causes a concentration-dependent quench in the fluorescence of Trp59 [37,39,40]. Using this assay, we could detect no difference in the affinity of rapamycin for any of the natively purified FKBP-12 proteins (Fig. 2E; Table 2). The mean equilibrium dissociation constants (Kd) varied minimally between 6 and 8 nM, in agreement with Ki values determined in the PPIase inhibition assays (Table 2) and literature values determined in similar fluorescence assays [39,40]. Refolded 6H-FKBP12 and cFKBP12 were again indistinguishable, both from each other and from their natively purified counterparts, in their ability to bind rapamycin. The mean Kd values for these two proteins are in the 6–9 nM range (Table 2). In contrast, the apparent mean Kd of refolded 6HLFKBP12 for rapamycin was 45 ± 7 nM; a 6-fold reduction in affinity (red line, Fig. 2F; Table 2). It is clear from these results that only refolded 6HL-FKBP12 has altered biochemical activities; the other five forms of FKBP-12 are indistinguishable from each other, and possess native activities. This altered biochemistry is a function of refolding; natively purified 6HL-FKBP12 has ‘‘native’’ activity in all of the assays performed. Additionally, the length of the his-tag appears to contribute to the aberrant activity of refolded 6HL-FKBP12; attaching a shorter his-tag to FKBP-12 has no effect on the proteins activity, natively purified or refolded following denaturation in urea. Similar experiments performed with different protein concentrations gave essentially identical results (data not shown). A simple explanation for the aberrant biochemistry of refolded 6HL-FKBP12 is that the addition of extra nonnative amino acid sequence results in two populations of protein molecules; one, natively folded and active and a second population which is misfolded and inactive. This mixed population would reduce the effective concentration of active protein. However, for this to be the case around

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25% of the refolded 6HL-FKBP12 would have to be totally inactive to account for the altered biochemical characteristics we observed for 6HL-FKBP12. In Fig. 2D, despite the lower intrinsic level of PPIase activity of refolded 6HLFKBP12 it takes relatively more rapamycin to get the same levels of inhibition compared to refolded 6H-FKBP12 and cFKBP12 (Fig. 2D). If there were less active protein— binding rapamycin with the same affinity as the other two ‘‘native’’ refolded forms—relatively more inhibition for the same amount of rapamycin would have been observed. To directly test this hypothesis, we analysed the binding of rapamycin to each of the refolded FKBP-12 proteins using isothermal titration calorimetry (ITC). ITC is particularly sensitive to variations in the active protein concentration and a preparation of protein containing a 20–25% inactive population would readily be detected in the value reported for the stoichiometry of binding. Typical titration curves are shown in Fig. 3. The best fit of the data (solid red lines, Fig. 3) was obtained with a single class of binding-site model. The mean Kd values for rapamycin binding to refolded cFKBP12, refolded 6H-FKBP12 and refolded 6HL-FKBP12 are 2 ± 0.5, 3.7 ± 1.2 and 25.2 ± 8.6 nM, respectively (Table 3). Refolded 6HLFKBP12 again shows a 6- and 7-fold reduction in affinity for rapamycin, while refolded cFKBP12 and 6H-FKBP12 exhibit native affinities, in good agreement with the PPIase and fluorescence data (Fig. 2; Table 2). Most interestingly however, the mean stoichiometry of binding rapamycin, for all three refolded FKBP12 forms, varied minimally around a molar ratio of protein to ligand of 0.95 (Table

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3). Thus, there appears to be, at most, 5% inactive protein in all three refolded preparations, providing good evidence that the altered biochemical activity of refolded 6HLFKBP12 is not a result of the presence of a significant population of grossly misfolded and inactive molecules. The entropic component for all three binding interactions (TDS, Table 3) is essentially the same 7 kcal mol1, with the loss of binding energy, for the rapamycin: 6HLFKBP12 interaction due mostly to a reduced (1.5 kcal mol1 less) enthalpy of binding (Table 3). These results suggest that the altered biochemistry of 6HLFKBP12 is better explained by a having a subtly misfolded protein that as a result has altered enzymatic activity and an altered physical interaction with rapamycin. N-terminal his-tags differentially reduce FKBP-12 stability FKBP-12 is readily reversibly denatured and refolded, via a two-step transition [21,23–27,41]. Therefore, we were interested to see if the longer fusion-tag affected the denaturation and refolding characteristics of human FKBP12. Natively purified 6H-FKBP12, 6HL-FKBP12 and cFKBP12 were subjected to urea-induced denaturation. The single tryptophan residue (Trp59) is highly sensitive to the folded conformation of the protein and its fluorescence is highly enhanced ( 6-fold) upon unfolding [21]. Typical titration curves are illustrated in Fig. 4 and all three FKBP-12 forms show a two-state unfolding transition under equilibrium conditions. Data from the titration curves were analysed to obtain values for [Urea]50% (the

Fig. 3. Calorimetric titration of refolded cFKBP12, 6H-FKBP12 and 6HL-FKBP12 binding to rapamycin. Each peak (top panel) represents the injection of 6 ll of 24.6 lM refolded cFKBP12 (A), 24.4 lM refolded 6H-FKBP12 (B) or 25.1 lM refolded 6HL-FKBP12 (C) into 1.33 lM rapamycin at 27 C in 50 mM Hepes, pH 8.0; 100 mM NaCl; and 1 mM NaN3. The enthalpy of binding (DH, kcal mol1 of injectant) was determined by integration of the injection peaks and correction for heats of dilution were determined in similar experiments minus rapamycin. The resulting binding isotherm (lower panel) was best fit (red lines) by a single class of binding site model with a 1:1 stoichiometry, using the MicroCal Origin software. Values for the appropriate thermodynamic parameters, determined from three repeated experiments, are shown in Table 3. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article).

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Table 3 Thermodynamic parameters for the binding of rapamycin to refolded cFKBP12, refolded 6H-FKBP12 and refolded 6HL-FKBP12 determined by isothermal titration calorimetry (ITC) Refolded protein

Kd (nM)

DHITC (kcal mol1)

TDSITC (kcal mol1)

Stoichiometry

DGITC (kcal mol1)

cFKBP12 6H-FKBP12 6HL-FKBP12

2.0 ± 0.5 3.7 ± 1.2 25.2 ± 8.6

19.07 ± 0.22 18.78 ± 0.26 17.31 ± 0.22

7.16 7.20 6.9

0.95 ± 0.02 0.96 ± 0.02 0.98 ± 0.03

11.91 ± 0.19 11.58 ± 0.23 10.41 ± 0.25

Experiments were performed at 27 C. Values shown, determined from experiments illustrated in Fig. 3, are the mean (±SE) from three repeated experiments with varying protein and rapamycin concentrations. The DHITC value for cFKBP12 is in good agreement with the value reported for wild type human FKBP-12; 19.33 ± 0.31 kcal mol1 [43]. TDSITC was calculated using the equations DGITC = DHITC  TDSITC and DGITC = RTlog10 1/Kd using the mean values for DHITC, DGITC and Kd.

Fig. 4. Addition of hexa-his tags of variable length differentially destabilizes FKBP-12 to urea denaturation. Typical urea induced denaturation curves (in 50 mM Tris, pH 7.5; 10 mM DTT) are shown for cFKBP12 (filled circles), 6H-FKBP12 (open triangles), 6HL-FKBP12 (open circles) and refolded 6HL-FKBP-12 (red filled diamonds). 0.62 lM protein was used in all experiments. Solid lines are the best fit of the data to Eq. (6) (see Materials and methods). Values calculated for m, [Urea]50% and DG(H2O) are shown in Table 4. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

concentration of urea causing 50% denaturation), m (a constant relating the increase in degree of exposure of the protein to the extent of denaturation), and DG(H2O) (the free energy of unfolding in water) (Table 4). 6HL-FKBP12 is destabilized compared to both cFKBP-12 and 6H-FKBP12. The [Urea]50% for 6HL-FKBP12 is 3.32 ± 0.06 M compared to 4 ± 0.05 M for cFKBP12 and 3.81 ± 0.04 M for 6H-FKBP12 (Table 4). 6H-FKBP12, although marginally destabilized compared to cFKBP12, is more stable than 6HL-FKBP12. The presence of the long his-tag destabi-

lized the protein by 0.84 kcal mol1, compared to the shorter his-tag, which destabilizes the protein by only 0.14 kcal mol1. The [Urea]50% and DG(H2O) values for cFKBP12 (4 M and 6.32 kcal mol1, respectively) agree with literature values for these constants for untagged human FKBP-12 (3.9 M and 6.2 kcal mol1, [Urea]50% and DG(H2O), respectively) [21,23,25]. Other workers have reported a reduced stability for a slightly different tetra his-tagged FKBP-12 fusion protein [27]. The [Urea]50% value for this protein was reduced from 3.9 M urea to 2.9 M urea [27]. In contrast, the equilibrium urea denaturation profile of refolded 6HL-FKBP-12 is altered from the natively purified form (red diamonds, Fig. 4). Refolded 6HL-FKBP12 is less stable than its natively purified counterpart; DG(H2O) is 4.57 ± 0.32 kcal mol1, a reduction in stability of 0.91 kcal mol1 (Table 4). The [Urea]50% was reduced very marginally from 3.32 ± 0.06 to 3.24 ± 0.07 M (Table 4). The m values for all three natively purified forms of the protein are nearly equivalent (1.58 ± 0.08, 1.6 ± 0.06 and 1.66 ± 0.07 kcal mol1 M1, cFKBP12 6HL-FKBP12 and 6H-FKBP12, respectively) indicating that denaturation occurs between similar thermodynamic states in all three proteins. However, the m value for refolded 6HLFKBP12 is considerably lower than the natively purified form (1.41 ± 0.07 kcal mol1 M1, Table 4). This indicates a less cooperative unfolding transition and/or a subtle change in the structure of the refolded 6HL-FKBP12, possibly due to miss incorporation of the longer fusion tag, somewhat, into the fold of the protein during the refolding process. The equilibrium urea denaturation profiles of refolded cFKBP-12 and refolded 6H-FKBP12 are essentially identical to their natively purified counter parts (data not shown).

Table 4 Free energies of unfolding for FKBP-12 proteins, determined by reversible urea denaturation experiments Protein species

DG(H2O) (kcal mol1)b

m(kcal mol1 M1)a

[Urea]50% (M)a

cFKBP-12 6H-FKBP-12 6HL-FKBP-12 Refolded 6HL-FKBP12

6.32 ± 0.4 6.18 ± 0.21 5.48 ± 0.38 4.57 ± 0.32

1.58 ± 0.08 1.6 ± 0.06 1.66 ± 0.07 1.41 ± 0.07

4.0 ± 0.05 3.81 ± 0.04 3.32 ± 0.06 3.24 ± 0.07

a b

Calculated for the fit of Eq. (6) to data in Fig. 4. Calculated using Eq. (5). Values are ± SE, calculated form the fitting program.

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The kinetics of refolding are slowest for 6HL-FKBP12 Alone, the loss of stability seems unlikely to account for the altered biochemistry of 6HL-FKBP12 in our other assays, as the natively purified 6HL-FKBP12 shows no difference in activity compared to either cFKBP12 or 6HFKBP12. The altered biochemistry of 6HL-FKBP12 only manifests itself after refolding of 6HL-FKBP12 in vitro. Thus, we surmised that the longer hexa-his fusion tag was interfering with the refolding pathway of FKBP-12 in vitro. We therefore investigated the refolding kinetics of cFKBP-12, 6H-FKBP12 and 6HL-FKBP12. The fluorescence of Trp59 is heavily quenched when FKBP-12 refolds [22,25,27]. This residue is buried in the core of the folded molecule in a highly hydrophobic environment at the base of the ligand binding pocket [18]. To initiate refolding, the corresponding denatured FKBP-12 protein was diluted 30-fold (0.2 M urea, pH 8.0), and refolding was followed by the quench in the intrinsic tryptophan fluorescence of the protein versus time. Representative data is shown in Fig. 5A. cFKBP12 refolds rapidly, whilst 6H-FKBP12 and 6HL-FKBP12 refold more slowly. 6HL-FKBP12 (red solid line) takes the longest to refold. For all concentrations of all three FKBP-12 proteins tested the final fluorescence intensity reached at steady state (>6000 s) was, within experimental error, the same (Fig. 5A). The folding of FKBP-12 occurs in three kinetic phases—a rapid initial phase (k1) followed by two slower phases [22,25,27]. The multiphasic nature of the refolding reaction results from the heterogeneous population of molecules in the unfolded state due to the isomerisation of the peptidyl–prolyl bonds in the seven proline residues present in the sequence of FKBP-12. The two slower phases (k2, k3) are limited by proline isomerisation [22,25,27]. Native FKBP-12 contains seven trans-prolyl peptide bonds. The rate of an auto-catalytic activity increases as a function of the concentration of FKBP-12 [27] and at concentrations above 4 lM autocatalysis proceeds very efficiently and likely represents the fast direct folding reaction of the protein molecules where all seven peptidyl–prolyl bond conformations are trans [25,27]. The fast phase (k1), the readily detectable in stop-flow experiments [22,25], is complete within the dead time (3–5 s) of the manual mixing experiments performed in this study. Similar experiments similar to those indicated in Fig. 5A were performed with different concentrations of protein, and the fluorescence signal fit to a double exponential function to give apparent rate constants for the slow isomerisation phases (k2 and k3). All three proteins show a concentration dependent acceleration in the slow proline isomerisation limited rates (Fig. 5B), although both his-tagged proteins were slower to refold than cFKBP12. 6HL-FKBP-12 was significantly slower to refold than 6H-FKBP12 (Fig. 5B). Similar observations have been reported for a tetra-his-tagged FKBP-12 [27]. This tetrahis-tagged FKBP-12 showed a greater acceleration in the refolding rate as a function of protein concentration than

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native FKBP-12. Increasing the concentration of this tetra-his-tagged protein from 0.05 to 10 lM resulted in a decrease in the refolding t1/2 from 270 to 24 s [27], similar to the behaviour of 6H-FKBP12 (Fig. 5B). The tetra-histagged FKBP-12 fusion [27] has a 16 amino acid tag (TMITNSMHHHHDDDDK); 6H-FKBP12 has a 13 amino acid tag (MRGSHHHHHHGSM). Full PPIase activity is regained by refolded cFKBP12 and 6H-FKBP-12 but not 6HL-FKBP12 We also analysed the kinetics of reactivation of the PPIase activity of refolding proteins. This was performed by removal of aliquots from the refolding solution, which was then incubated for 10 s in the PPIase assay mixture in the presence of a-chymotrypsin to degrade unfolded and partially folded molecules and the PPIase activity then assayed ([27] and see materials and methods. The % relative PPIase activity, compared to the activity of the natively purified form of the same protein assayed in a similar manner, was then plotted versus time. Representative results are shown in Fig. 6. Again, cFKBP-12 recovers activity quickly, 6H-FKBP12 is slower to recover and 6HLFKBP-12 is slower still (Fig. 6A). This trend was observed at several different protein concentrations (data not shown). At equilibrium (>6000 s) both refolded 6HFKBP12 and refolded cFKBP12 regain, within experimental error, full native PPIase activity; 96.8 ± 2.3% and 95.7 ± 1.9%, 6H-FKBP12 and cFKBP12, respectively (mean ± SD). In contrast, refolded 6HL-FKBP12 only recovered 83.2 ± 2% (mean ± SD) of native activity (Fig. 6B). Gross misfolding events do not explain the altered activity of refolded 6HL-FKBP12 As already noted above, a simple explanation for the aberrant biochemistry of refolded 6HL-FKBP12 is that the long fusion tag results in two populations of protein molecules; one, natively folded and active, the second, completely misfolded and inactive. The purified refolded protein preparations used in the biochemical analysis were highly monomeric species; analytical gel filtration showed all three refolded FKBP-12 proteins eluted as single peaks with molecular masses very close to their predicted masses (Fig. 1C). Our ITC data showed that all three refolded forms of the protein bound rapamycin, within experimental error, with a 1:1 molar stoichiometry (Fig. 3, Table 3). This data provide good evidence that there is little misfolded and inactive protein in the refolded 6HL-FKBP12 preparations. Furthermore, the data in Figs. 2E, 2F, 4 and 5A show the finite fluorescence intensities of all three proteins at the same concentration have very similar values (±3%), both at the beginning and end of each particular experiment. Given the high sensitivity of the fluorescence of Trp59 to its environment in terms of the folded conformation of FKBP-12 [21,25,27], this further

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Fig. 5. Hexa-his tags slow the refolding kinetics of FKBP-12. (A) The refolding 0.31 lM FKBP-12 is monitored by the quench in the fluorescence intensity (a.u.) of Trp59 at 354 nm versus time. Refolding was initiated by diluting the unfolded protein 1:30 (in 50 mM Hepes, pH 8.0; 10 mM DTT; and 6 M urea) to 0.2 M urea in the same buffer, at 4 C. (B) Plot the final protein concentration in the refolding reaction versus the apparent rate constants (k2 and k3, filled circles and open circles, respectively) for the slow proline isomerisation refolding phases. These parameters were determined by fitting a double exponential function to data similar to that shown in (A).

suggests that the overall folded conformations of all three of the refolded FKBP-12 proteins are very similar. The m value for refolded 6HL-FKBP12 determined in equilibrium urea denaturation experiments is considerably lower than the m value of the natively purified form. This is indicative of a less cooperative unfolding transition and/ or a subtle change in the structure protein. Additionally, thrombin cleavage was less efficient with refolded 6HLFKBP12 than with natively purified protein (data not

shown), suggesting that the protease recognition sequence is less accessible in this protein presumably as a result of the longer tag being incorporated into a misfolded form of the protein. We did not observe any time-dependent aggregation or formation of precipitate in refolded preparations of 6HL-FKBP12 (data not shown). Our data fits with a model in which the longer fusion-tag becomes incorporated into the structure of 6HL-FKBP12 upon refolding in vitro of protein, producing a stable but

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Fig. 6. Reactivation of PPIase activity of refolded FKBP-12. (A) Refolding was initiated by diluting the unfolded protein 1:30 (in 50 mM Hepes, pH 8.0; 10 mM DTT; and 6 M urea) to 0.2 M urea in the same buffer, at 4 C. The data shown are for a final protein concentration of 0.31 lM. Assaying the recovery of PPIase activity was performed by taking aliquots were from the refolding solution, incubated for 10 s in the PPIase assay mixture in the presence of a-chymotrypsin to degrade unfolded and partially folded molecules and the PPIase activity assayed (see Materials and methods). The % relative PPIase activity, compared to the activity of the native form of the same protein assayed in a similar manner, is plotted versus time. (B) Graphical representation showing the final % relative PPIase activity regained at equilibrium ( 6,000 s) after refolding for several protein concentrations. Refolded 6HL-FKBP12 only recovered 83.2 ± 2% (mean, ±SD) of native activity, compared to 96.8 ± 2.3% and 95.7 ± 1.9% (mean, ±SD) activity for refolded 6HFKBP12 and cFKBP12, respectively.

kinetically trapped [8] form of the protein with subtly altered biochemistry. Very subtle structural differences between FKBP-12 and its isoform FKBP-12.6 [42] are almost entirely attributed to the residue at position 59 (tryptophan in FKBP-12, phenylanine in FKBP-12.6) [26], which sits at the base of the hydrophobic substrate binding pocket. These two isoforms also show selectivity in terms of their binding specificity to ryanodine receptors; FKBP-12 binds to the ryanodine receptor in skeletal muscle, FKBP-12.6 binds the ryanodine recptor in cardiac muscle [42]. Mutation of Trp59 to either Phe or Leu in FKBP-12 results in a large stabilization (2.72 and 2.35 kcal mol1, respectively [26] of the protein and slight rearrangement of secondary structure elements around the binding site [26]. Furthermore, an extensive

context-dependent mutational analysis of the contribution of individual amino acids to the stability of FKBP-12 showed that single point mutations, in residues remote from the binding/catalytic site very significantly altered the stability of the protein fold [23,26]. One might expect misfolding events and the generation of misfolded intermediates to be more prevalent as one adds increasing lengths of essentially random sequence to a small protein. Indeed, the shorter tag (13 amino acids, MRGSHHHHHHGSM) slows refolding, but does not interfere with the final biochemical activities; the highly polar nature of the poly-histidine portion of the tag is likely to prevent the miss-incorporation of this short sequence into the fold of a small protein like FKBP-12 [1]. Similar increases in the refolding time and recovery of native PPI-

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ase activity levels were also observed for a tetra-his-tagged FKBP-12 (this construct has a 16 amino acid tag) [27]. However, the longer tag, 20 amino acids in length (MGSSHHHHHHSSGLVPRSHM) and nearly one fifth of the length of native FKBP-12, not only further slows the rate of refolding, but also causes altered biochemical characteristics. The sequence composition of the tag may also play a role in the slower refolding rates of 6HL-FKBP12. FKBP-12 has distinct preferences for the amino acid immediately preceding the proline residue in terms of the efficiency of the isomerase activity [35]. The longer tag contains an additional proline residue preceded by a valine residue. FKBP-12 catalyses the cis–trans isomerisation of the peptidyl–prolyl bond of Val-Pro with an 80% reduced efficiency compared to that of Leu-Pro [35]. The existence of an additional inefficiently isomerised peptidyl–prolyl bond in the longer tag may help explain the markedly slower rates of refolding for 6HL-FKBP12 following urea denaturation. In addition, as refolding 6HL-FKBP12 in vitro appears to result in the formation of a protein that is a 20% less efficient enzyme, and one that binds ligands with 6-fold reduced affinity, this would likely reduce the proteins ability to facilitate the auto-catalysis of its own refolding. This provides an explanation for the lower concentration-dependent acceleration observed for 6HL-FKBP12s refolding rates compared to 6H-FKBP12 and cFKBP12 (Fig. 5). Korepanova et al [41] have reported that even very short N-terminal extensions (three amino acids in length) can effect not only the equilibrium stability of FKBP-12 but also affect the kinetics of unfolding and refolding of the protein. Equilibrium urea denaturation experiments demonstrated that FKBP-12 containing an N-terminal 3 amino acid extension, Gly-Ser-Met, was destabilized by 0.49 kcal mol1, compared to FKBP-12 with no N-terminal extension, while one with a Gly-Ser-Gly N-terminal extension was destabilized by 1.75 kcal mol1 [41]. NMR studies indicated that the N-terminal extensions perturbed the protein structure near the N-terminus, likely explaining the loss of stability [41]. However, structural perturbations were shown to have been propagated into secondary structural elements contributing to the proteins ligand binding site [41]. Additionally, stop-flow experiments performed with these two forms of FKBP-12 suggested the formation of a kinetic folding intermediate not observed in the folding kinetics wild type FKBP12 [41]. Furthermore, different amino acid sequences in the extension contribute differentially to the observed equilibrium and kinetic effects [41]. It has been suggested that the N terminus plays important role for the in the folding of FKBP—especially so as the folding pathway for FKBP-12 involves a loop crossing event [17], that may be inhibited or altered due to the presence of the extra non-native sequence. The severity of the destabilization and alteration the kinetics of folding appears to be differentially affected by the sequence of the N-terminal extension; our long his-tag may be particularly detrimental to the stability and refolding of FKBP-12.

From other sets of stop-flow kinetic experiments there is evidence that the ligand binding site is only very weakly formed in the rapidly generated transition state in FKBP12s folding pathway [25]. The presence of an extra 20 non-native amino acids on the N-terminus of FKBP-12 may inhibit the proper structural rearrangements that allow native FKBP-12 to fold correctly into and/or from this transition state. Concluding remarks Our data fits with a model in which the introduction of extra non-native sequence into the folding pathway of a small protein like FKBP-12 becomes—to a lesser or greater degree—incorporated into the protein, resulting in destabilization and loss of activity. The effects exhibited by refolded 6HL-FKBP12 are subtle but potentially important. If a 6-fold loss of affinity was seen with a well defined and naturally tight binding partner—in this case rapamycin— a similar or even greater loss of affinity for a potential lead compound in a screen for new inhibitors might result in such compounds not being picked up as a Kd of 10 lM would be weakened to around 100 lM. We have clearly shown that only refolded 6HL-FKBP12 exhibits altered biochemistry. The protein purified under native conditions showed native activity, suggesting that the folding/chaperone machinery in the cell very effectively precludes formation of any subtly miss-folded, kinetically trapped intermediates [8] and it is likely that refolding purified recombinant proteins with longer his-tags may frequently result in folded forms that differ subtly from the native fold produced within the bacterial cell. Acknowledgments We thank members of the Structural Biochemistry Group for helpful discussions. The work was supported by the MRC, The Welcome Trust, and the Edinburgh Protein Interaction Centre (EPIC). References [1] K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 60 (2003) 523–533. [2] D.S. Waugh, Making the most of affinity tags, Trends Biotechnol. 23 (2005) 316–320. [3] I. Hunt, From gene to protein: a review of new and enabling technologies for multi-parallel protein expression, Protein Expr. Purif. 40 (2005) 1–22. [4] S.A. Doyle, Screening for the expression of soluble recombinant protein in Escherichia coli, Methods Mol. Biol. 310 (2005) 115–121. [5] S.A. Doyle, High-throughput cloning for proteomics research, Methods Mol. Biol. 310 (2005) 107–113. [6] J. Wu, M. Filutowicz, Hexahistidine (His6)-tag dependent protein dimerization: a cautionary tale, Acta Biochim. Pol. 46 (1999) 591– 599. [7] C.M. Halliwell, G. Morgan, C.P. Ou, A.E. Cass, Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules, Anal Biochem. 295 (2001) 257–261.

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