Báo cáo khoa học: Isothermal unfolding studies on the apo and holo forms of Plasmodium falciparum acyl carrier protein Role of the 4¢-phosphopantetheine group in the stability of the holo form of Plasmodium falciparum acyl carrier protein docx

Isothermal unfolding studies on the apo and holo forms of Plasmodium falciparum acyl carrier protein Role of the 4¢-phosphopantetheine group in the stability of the holo form of Plasmodium falciparum acyl carrier protein Rahul Modak1, Sharmistha Sinha2 and Namita Surolia1 Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India Keywords apo-ACP; conformational stability; holo-ACP; isothermal unfolding; 4¢-phosphopantetheine Correspondence N Surolia, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India Fax: +91 80 22082766 Tel: +91 80 2208282021 E-mail: surolia@jncasr.ac.in (Received 29 January 2007, revised 15 April 2007, accepted May 2007) doi:10.1111/j.1742-4658.2007.05856.x The unfolding pathways of the two forms of Plasmodium falciparum acyl carrier protein, the apo and holo forms, were determined by guanidine hydrochloride-induced denaturation Both the apo form and the holo form displayed a reversible two-state unfolding mechanism The analysis of isothermal denaturation data provides values for the conformational stability of the two proteins Although both forms have the same amino acid sequence, and they have similar secondary structures, it was found that the – DG of unfolding of the holo form was lower than that of the apo form at all the temperatures at which the experiments were done The higher stability of the holo form can be attributed to the number of favorable contacts that the 4¢-phosphopantetheine group makes with the surface residues by virtue of a number of hydrogen bonds Furthermore, there are several hydrophobic interactions with 4¢-phosphopantetheine that firmly maintain the structure of the holo form We show here for the first time that the interactions between 4¢-phosphopantetheine and the polypeptide backbone of acyl carrier protein stabilize the protein As Plasmodium acyl carrier protein has a similar secondary structure to the other acyl carrier proteins and acyl carrier protein-like domains, the detailed biophysical characterization of Plasmodium acyl carrier protein can serve as a prototype for the analysis of the conformational stability of other acyl carrier proteins Malaria continues to exact the highest mortality and morbidity rate after tuberculosis ‘The scourge of the tropics’, malaria is endemic in 100 countries in the world Approximately 500 million cases of malaria are reported every year, and 3000 children die of malaria every day [1] Our recent demonstration of the occurrence of the type II fatty acid synthesis (FAS) pathway in the malaria parasite, Plasmodium falciparum, and its inhibition by triclosan, an inhibitor of the rate- determining enzyme of type II FAS, enoyl-acyl carrier protein (ACP) reductase, proved the pivotal role played by this pathway in the survival of the malarial parasite The essential role of fatty acids and lipids in cell growth and differentiation, and the occurrence of a different type (type I) of fatty acid biosynthetic pathway in the human host from that of the malaria parasite, make this pathway an attractive target for developing antimalarial agents [2,3] Abbreviations AAS, acyl-ACP synthase; ACP, acyl carrier protein; AcpS, holo-ACP synthase; apo-ACP, Plasmodium falciparum acyl carrier protein (apo form); FAS, fatty acid synthesis; holo-ACP, Plasmodium falciparum acyl carrier protein (holo form); holo-ACP, acyl carrier protein (holo form); LEM, linear extrapolation model; 4¢-PP, 4¢-phosphopantetheine; PfACP, Plasmodium falciparum acyl carrier protein (both apo and halo forms) FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3313 Plasmodium falciparum acyl carrier protein R Modak et al The type II FAS pathway, found in most bacteria, plants and the malaria parasite, consists of distinct enzymes, each catalyzing individual reactions required to complete successive cycles of fatty acid elongation, in contrast to the multifunctional enzyme catalyzing all the steps of the type I FAS pathway [4,5] ACP is an essential component of both type I and type II fatty acid synthesis pathways Whereas in the type I FAS pathway, it is an integral part of the multifunctional enzyme, it is a discrete entity shuttling acyl groups to the successive enzymes in the type II FAS pathway ACP is a small protein of molecular mass 8–10 kDa It plays essential roles in a myriad of metabolic pathways Assorted functions involve fatty acid and lipid biosynthesis, lipid A formation, membrane-derived oligosaccharide biosynthesis, and activation of RTX (repeats in toxin), toxins of Gram-negative bacteria [6– 13] In particular instances, specialized ACPs operate in restricted pathways such as rhizobial nodulation signaling, and polyketide and lipoteichoic acid synthesis [11,12] ACP plays a pivotal role in fatty acid synthesis as well as in its utilization It carries the growing acyl chain from one enzyme of the FAS pathway to the other in a sequential manner Given its crucial roles in metabolism, the high degree of conservation of ACP’s primary structure is not surprising The three-dimensional structure of Escherichia coli ACP is the prototype of bacterial and plant ACP structures [14–17] The solution structure of ACPs consists of a threehelix bundle and a short fourth helix, all connected by loops, with a long, structured turn between helices I and II ACP in its holo form exists in a dynamic equilibrium between the two conformers [14–22] ACP is synthesized as an apoprotein (apo-ACP) and undergoes post-translational modification by holo-ACP synthase Holo-ACP synthase transfers the 4¢-phosphopantetheine (4¢-PP) group from CoA to a particular serine residue of apo-ACP (Ser37 in PfACP) The growing fatty acid chain is attached to the terminal sulfhydryl group of the phosphopantetheine, the only sulfhydryl group in most ACPs All known ACPs (or ACP-like domains) undergo this modification, and all share sequence similarities around the modified serine [22] PfACP is a protein of 137 residues, inclusive of signal and transit sequences, required for targeting of the protein to the apicoplast The mature protein comprises 79 amino acids (residues 58–137) [23] Recently, the solution structure of P falciparum holo-ACP Fig (A) PfACP expression: SDS ⁄ PAGE (15%) showing the elution profile of PfACP with N-terminal His-tag Lane 1: supernatant of isopropyl-b-D-thiogalactopyranoside-induced E coli cultures transformed with pET-28a(+)-ACP Lane 2: protein markers; the protein bands correspond to 116 kDa, 66.2 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, and 14.4 kDa (from top to bottom) Lanes 3–7: different fractions of PfACP eluted at 50 mM imidazole (B) PfACP expression: native PAGE (12%) showing the ratio of holo-ACP and apo-ACP in the eluted fractions from an Ni–nitrilotriacetic acid agarose column Lanes 1–3: different fractions of PfACP eluted at 50 mM imidazole (C) Size exclusion chromatography profile of PfACP: holo-ACP dimer has been separated from a mixture of apo-ACP and holo-ACP monomers by size exclusion chromatography using a Superdex 75 column (30 cm) equilibrated and eluted with 20 mM Tris (pH 6.5) and 200 mM NaCl Peak 1: holo-ACP dimer Peak 2: mixture of apo-ACP and holo-ACP monomers (D) Separation profile of holo-ACP dimer and apo-ACP and holo-ACP monomers: 12% native PAGE showing the separation of holo-ACP dimer from a mixture of apo-ACP and holo-ACP monomers Lane 1: holo-ACP dimer without dithiothreitol Lane 2: holo-ACP dimer with dithiothreitol Lane 3: mixture of apo-ACP and holo-ACP monomers (E) Removal of His-tag from recombinant PfACP For the cleavage of His-tag, unit of thrombin was used for mg of PfACP at 25 °C for h On 12% native PAGE, ACPs with and without His-tag showed significant differences in mobility Lane 1: holo-ACP with His-tag Lane 2: holo-ACP without His-tag Lane 3: mixture of holo-ACP monomer and apo-ACP with His-tag Lane 4: mixture of holo-ACP monomer and apo-ACP without His-tag (F) Separation of apo-ACP and holo-ACP by anion exchange chromatography Elution profile of apo-ACP and holo-ACP on a MonoQ HR ⁄ anion exchange column Peak 1: apo-ACP Peak 2: holo-ACP (G) Separation of apo-ACP and holo-ACP; 12% native PAGE showing the separation of apo-ACP and holo-ACP by anion exchange chromatography Lane 1: mixture of apo-ACP and holo-ACP Lane 2: purified apo-ACP Lane 3: purified holo-ACP (H) Dynamic light-scattering data of PfACP (a) Particle size distribution of apo-ACP The solid lines indicate the accumulation percentages of particles (b) Particle size distribution of holo-ACP The solid lines indicate the accumulation percentages of particles (I) Sucrose density gradient sedimentation analysis Forty micrograms of apo-ACP and holo-ACP were layered on top of a mL continuous 0–10% (w ⁄ v) sucrose density gradient, and this was followed by centrifugation, fractionation and 12% native PAGE, as described in Experimental procedures Protein bands were visualized by silver staining (a) Lane 1: apo-ACP Lane 2: holo-ACP Lanes 3–9: fractions 18–12 of sucrose density gradient for apo-ACP Lanes 10–15: fractions 18–13 of sucrose density gradient for holo-ACP (b) Lanes 1, and 3, respectively, are fractions 16–18 of sucrose density gradient for holo-ACP under oxidizing conditions (c) Apo-ACP (O) and holo-ACP (h) in each fraction was quantified by measuring the intensity of the silver-stained protein bands using QUANTITY ONE software and plotted against the fraction number (AU, arbitrary unit) (d) The apparent molecular masses of apo-ACP and holo-ACP were estimated on the basis of the linear regression of the fraction number of the molecular mass markers cytochrome c (CyC), carbonic anhydrase (CA), and BSA From the calibration curve of the sucrose density gradient, the estimated molecular masses of apo-ACP, holo-ACP monomer and holoACP dimer are $ 16.75 kDa, 21 kDa and 26.5 kDa, respectively 3314 FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS R Modak et al Plasmodium falciparum acyl carrier protein (holo-ACP) has been solved by Sharma et al and it is found to exist in conformational equilibrium between the two states [24,25] These two states have been identified as the major and the minor forms of the holoACP structure, on the basis of their percentage contributions (65% and 35%, respectively) to the overall structure of the protein The structures of the major and minor conformations of holo-ACP bear close resemblance to that of E coli butyryl-ACP, with rmsd ˚ ˚ values of 2.24 A and 2.19 A, when superimposed on their backbone atoms In the present study, we report the detailed biophysical characterization of both apo-ACP and holo-ACP to ascertain their conformational stabilities An interesting outcome of the study, reported for the first time, Results Expression and purification of ACP The mature PfACP (without the signal and transit sequence) was expressed in E coli BL21 (DE3) cells with an N-terminal His-tag PfACP was purified by Ni–nitrilotriacetic acid agarose affinity chromatography to homogeneity, as shown in Fig 1A The purified protein on 15% SDS ⁄ PAGE gel has a monomeric A280 (mAU) B A 116 66.2 45 for this large family of essential proteins, is that the 4¢-PP prosthetic group imparts considerably higher conformational stability (– DG) to holo-ACP as compared to its apo-ACP counterpart Holo-ACP dimer 35 Apo-ACP Holo-ACP 25 C D Holo-ACP dimer Apo-ACP Holo-ACP 18.4 Retention time (min) 14.4 1 Apo-ACP with his-tag Holo-ACP with his-tag F G A280(mAU) E Apo-ACP Holo-ACP Retention time (min) Apo-ACP Holo-ACP 1 I-c Fraction no Band intensity (A.U.) I-b H-b H-a 1800 1600 1400 1200 1000 800 600 400 200 10 I-a 12 14 Fraction no 16 18 18 16 14 12 10 1.0 CyC Apo 10 11 12 13 14 15 I-d Holo CA BSA 1.2 1.4 1.6 Log (M.W.) 1.8 2.0 FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3315 Plasmodium falciparum acyl carrier protein R Modak et al molecular mass of $ kDa The ratio of holo-ACP and apo-ACP was determined to be in the range of ±50% by 12% native PAGE (Fig 1B) Heterologously expressed PfACP is partly converted to holo-ACP by E coli holo-ACP synthase Holo-ACP forms a disulfide-bonded dimer through the thiol group of phosphopantetheine in a nonreducing environment The holo-ACP dimer was separated from the mixture of apo-ACP and holo-ACP monomers by size exclusion chromatography under nonreducing conditions (Fig 1C,D) From the calibration curve for the Superdex 75 column, with standard globular proteins, the apparent molecular mass of holo-ACP dimer and the mixture of holo-ACP monomer and apo-ACP were found to be 33 kDa and 25 kDa, respectively Purified holo-ACP and the mixture of apo-ACP and holo-ACP monomer were subjected to thrombin cleavage to remove the histidine tag from the protein Approximately 90% ACP cleavage was achieved, and uncleaved ACP was removed by passage through an Ni–nitrilotriacetic acid affinity column (Fig 1E) ApoACP and holo-ACP monomers from the mixture were purified by anion exchange chromatography using a Mono Q HR ⁄ column (Fig 1F,G) The elution profile (Fig 1F) shows that apo-ACP has weaker affinity and eluted with 190 mm NaCl, whereas holo-ACP was eluted with 200 mm NaCl MALDI-TOF MS yielded molecular masses of 9418.845 Da (calculated 9417.65 Da) and 9752.831 Da (calculated 9751.65 Da) for apo-ACP and holo-ACP, respectively [Figs 2Aa,b] Dynamic light-scattering studies of PfACP Apo-ACP and holo-ACP yielded hydrodynamic radii of 1.95 ± 0.05 nm and 1.9 ± 0.1 nm, respectively, confirming that they have a single species over the entire experimental concentration range [Fig 1Ha,b] Sucrose density gradient sedimentation In sucrose density gradient sedimentation experiments, both apo-ACP and holo-ACP were detected between fractions 12 and 18 [Fig 1Ia–d]; apo-ACP showed a major peak in fraction 15, whereas holo-ACP showed a peak in fraction 14 [Fig 1Ia–d] From the calibration curve of the sucrose density gradient, the estimated molecular masses of apo-ACP and holo-ACP monomers are $ 16.75 kDa and 21 kDa, respectively The dimeric peak of holo-ACP was found in major amounts in fraction 17 when sucrose density gradient sedimentation for holo-ACP under oxidizing conditions was performed Fig (A) Molecular mass determination of apo-ACP and holo-ACP Molecular masses of apo-ACP and holo-ACP were determined with an Ultra Flex TOF ⁄ MALDI-TOF mass spectrometer (a) Mass spectrum of holo-ACP single major peak (9752.83 Da) [holo-ACP (calculated 9751.65 Da)] (b) Mass spectrum of apo-ACP, showing a single major peak of molecular mass 9418.84 Da [apo-ACP (calculated 9417.65 Da)] (B) Secondary structure of ACP The secondary structures of both apo-ACP (O) and holo-ACP (h) were determined by far-UV CD spectroscopy CD spectra show the presence of only a-helices as the secondary structure element in both apo-ACP and holo-ACP (C) Guanidine hydrochloride-induced transitions for holo-ACP (.) and apo-ACP (O) at 30 °C as monitored by CD at 222 nm The proteins were in buffer containing mM NaCl ⁄ Pi, 100 mM NaCl and mM dithiothreitol, plus the indicated concentration of guanidine hydrochloride The solid lines indicate the best-fit values for each curve (D) Refolding of ACP Far-UV CD spectra of native apo-ACP (d) and holo-ACP (.), and refolded apo-ACP (O) and holo-ACP (,) The CD spectra of native and refolded PfACP overlap, which shows that isothermal denaturation of PfACP is completely reversible (E) Fluorescence spectra of ACP at 25 °C The samples were excited at 280 nm, and emission spectra were recorded from 295 nm to 350 nm No change of emission maxima from 305 nm was observed for denatured holo-ACP (,) and apo-ACP (d), and native holo-ACP (.) and apo-ACP (O) For both forms, the only change in fluorescence intensity was observed upon denaturation (F) Refolding of ACP Both apo-ACP and holo-ACP denatured in M guanidine hydrochloride were refolded by dialysis (4 · 1000 mL) against mM Na ⁄ K phosphate (pH 6.5), 100 mM NaCl and mM dithiothreitol Both apo-ACP and holo-ACP are completely refolded after complete denaturation with M guanidine hydrochloride, as revealed by the equal mobilities of the native and refolded proteins on 12% native PAGE Lane 1: native apo-ACP Lane 2: refolded apo-ACP Lane 3: native holo-ACP Lane 4: refolded holo-ACP There is no degradation of holo-ACP to apo-ACP during the refolding process (G) AcpS assay Both native and refolded apo-ACPs were used as substrates for the AcpS assay, to convert them to lauroyl-ACP The reaction mixtures are checked on 12% native PAGE Lane 1: refolded apo-ACP Lane 2: refolded holo-ACP Lane 3: reaction mixture with native apo-ACP Lane 4: reaction mixture with refolded apo-ACP (H) AAS assay Both native and refolded holo-ACPs were used as substrates for AcpS assay, to convert them to lauroyl-ACP The reaction mixtures were checked on 20% conformation-sensitive PAGE with M urea Lane 1: refolded holo-ACP Lane 2: reaction mixture with refolded holo-ACP Lane 3: reaction mixture with native holo-ACP (I) Confirmation of AcpS reaction product The molecular masses of apo-ACP and holo-ACP were determined with an Ultra Flex TOF ⁄ MALDI-TOF mass spectrometer (a) Mass spectrum of refolded apo-ACP, showing a single major peak of molecular mass 9420.639 Da [apo-ACP (calculated 9417.65 Da)] (b) Mass spectrum of reaction mixture with native apo-ACP, showing a single major peak of molecular mass 9943.976 (Da) [lauroyl-ACP (calculated 9935 Da)] (c) Mass spectrum of reaction mixture with refolded apo-PfACP, showing a single major peak of molecular mass 9941.941 (Da) [lauroyl-ACP (calculated 9935 Da)] (d) Mass spectrum of refolded holo-ACP, showing a single major peak (9759.106 Da) [holo-ACP (calculated 9751.65 Da)] 3316 FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS R Modak et al Plasmodium falciparum acyl carrier protein 4.0e+5 a A 2.0e+5 B 0.0 b Molar ellipticity –2.0e+5 –4.0e+5 –6.0e+5 –8.0e+5 –1.0e+6 –1.2e+6 –1.4e+6 –1.6e+6 –1.8e+6 190 200 210 220 230 240 250 260 Wavelength (nm) 0.0 C D Molar ellipticity fraction native –5.0e+5 –1.0e+6 –1.5e+6 –2.0e+6 –2.5e+6 200 220 210 230 240 250 Wavelength (nm) [GdnHCI] M 4.5e+6 4.0e+6 F E G Fluorescence 3.5e+6 3.0e+6 2.5e+6 2.0e+6 1.5e+6 1.0e+6 5.0e+5 290 300 310 320 330 340 350 360 Wavelength (nm) 9920.639 H I a 9943.938 b holoACP 9941.941 c d 9758.108 C12-ACP FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3317 Plasmodium falciparum acyl carrier protein R Modak et al cence coincided well for both apo-ACP and holo-ACP, indicating that their denaturation process is a two-state reaction (Fig 3A,B) Several representative guanidine hydrochloride-dependent denaturation experiments were done in the range 15°) 60 °C The results of the analysis of these curves using the linear extrapolation model (LEM) are shown in Tables and and Fig 4A–C There is a slight temperature dependence of the Cm value (Fig 4A), but the m value is independent of temperature, as per the expectations of LEM (Fig 4C) The mean m values for the apo form and the holo form are ) 1.64 kcalỈmol)1Ỉm)1 and ) 1.97 kcalỈmol)1Ỉm)1, respectively The DGwater values showed strong temperature dependence, with maximum stability at 30 °C (Fig 4B) Unfolding experiments were also monitored by the change in fluorescence anisotropy of the single tyrosine residue at the C-terminus of ACP The isothermal denaturation probed by fluorescence anisotropy correlated well with the far-UV CD and fluorescence quenching studies for both apo-ACP and holo-ACP, further indicating that their denaturation process is a two-state reaction (Fig 3C) These data further indicate that apo-ACP has lower stability than holoACP Biophysical studies with ACP The conformations of both apo-ACP and holo-ACP at pH 6.5 have been determined by far-UV CD spectroscopy Wavelength scans from 190 nm to 250 nm show that both forms of ACP have predominantly a-helices, which is in accordance with known ACP structures (Fig 2B) [24] The conformational stability of holo-ACP and apoACP was determined by chaotrope-dependent unfolding at different temperatures The reversibility of the isothermal denaturation of ACP was shown by the return of CD and fluorescence signals upon refolding after complete denaturation with m guanidine hydrochloride (Fig 2D,E) It was also found that the refolded apo-ACP and holo-ACP have mobilities comparable to that of the nondenatured wild-type counterparts on 12% native PAGE, which further confirms the reversibility of the transition (Fig 2F) Unfolding experiments monitored by the change in mean residue ellipticity at 222 nm [h]222 demonstrated that both forms undergo a two-state unfolding transition (Fig 2C) Unfolding transitions were also monitored by following the tyrosine fluorescence at 305 nm upon excitation at 280 nm This was done because PfACP is devoid of tryptophan but contains one tyrosine residue Fluorescence emission spectra of fully denatured PfACPs showed no shift in their emission maxima from 305 nm, but there was a substantial increase in fluorescence intensity (Fig 2E) The isothermal denaturation probed both by the far-UV CD and fluores- 0.8 Fluorescence (Fit) Far UV-CD (Fit) Fluorescence (Expt) Far UV-CD (Expt) 1.0 0.6 0.4 0.2 0.0 0.8 0.6 0.4 0.2 0.0 GdnCI concentration [M] Fraction native 0 GdnCI concentration [M] C [GdnHCI] M 3318 E coli holo-ACP synthase (AcpS) has been cloned and expressed in the laboratory as a His-tagged protein B Fluorescence (Fit) Fluorescence (Expt) Far UV-CD (Fit) Far UV-CD (Expt) 1.0 Fraction native Fraction native A Acyl-ACP synthesis assay with apo-ACP 7 Fig Comparison of guanidine hydrochloride-induced transitions (A) Comparison of guanidine hydrochloride-induced transitions of apo-ACP at 30 °C as monitored by far-UV CD at 222 nm (d) and tyrosine fluorescence at 305 nm (O) (B) Comparison of guanidine hydrochloride-induced transitions of holo-ACP at 30 °C monitored by CD at 222 nm (m) and tyrosine fluorescence at 305 nm (n) (C) Comparison of guanidine hydrochloride-induced transitions of apo-ACP (O) and holo-ACP (d) at 30 °C monitored by fluorescence anisotropy of tyrosine fluorescence at 305 nm FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS R Modak et al Plasmodium falciparum acyl carrier protein Table Parameters obtained from the fit of isothermal unfolding data by fitting Eqs (1)–(5) ND, not determined Temperature (K) Cm holo-ACP (M) Cm apo-ACP (M) m-value holo-ACP (kcalỈmol)1ỈM)1) m-value apo-ACP (kcalỈmol)1ỈM)1) DGwater holo-ACP (kcalỈmol)1) DGwater apo-ACP (kcalỈmol)1) 283 288 293 298 303 313 318 323 328 333 3.95 3.91 3.88 3.83 3.92 3.62 3.68 3.74 3.61 3.58 3.56 3.48 3.46 3.37 3.59 3.35 3.49 ND 3.31 3.27 ) ) ) ) ) ) ) ) ) ) ) 1.31 ) 1.90 ) 1.92 ) 1.40 ) 1.42 ) 1.51 ) 1.43 ND ) 2.17 ) 1.70 2.60 3.09 3.17 3.12 3.57 2.71 2.28 2.04 1.86 1.01 2.19 2.51 2.89 2.78 2.99 2.38 1.99 ND 1.08 0.74 ± ± ± ± ± ± ± ± ± ± 0.07 0.14 0.04 0.04 0.06 0.14 0.07 0.17 0.04 0.07 ± ± ± ± ± ± ± 0.06 0.07 0.09 0.06 0.05 0.12 0.05 ± 0.09 ± 0.05 1.55 2.12 3.12 2.37 1.67 1.95 1.75 1.87 1.97 1.67 ± ± ± ± ± ± ± ± ± ± Average Cm (M) Average m (kcalỈmol)1ỈM)1) Apo-ACP Holo-ACP 3.43 ± 0.04 3.77 ± 0.05 ± ± ± ± ± ± ± 0.25 0.37 0.43 0.14 0.37 0.37 0.08 ± 0.37 ± 0.37 refolded apo-ACP are equally and quantitatively converted to lauroyl-ACP (Fig 2G,I) Table Average Cm and m for apo-ACP and holo-ACP in the experimental temperature range Protein 0.18 0.07 0.55 0.31 0.17 0.62 0.78 0.65 0.65 0.21 ) 1.64 ± 0.12 ) 1.97 ± 0.09 Acyl-ACP synthesis assay with holo-ACP E coli acyl-ACP synthase (AAS) utilizes holo-ACP as a substrate and converts it to acyl-ACP E coli AAS also utilizes fatty acids with various chain lengths as substrates, producing the corresponding acyl-ACPs This property of AAS was utilized to check the correct refolding of holo-ACP An acyl-ACP synthesis assay clearly showed that both native and refolded holoACP are partially converted to lauroyl-ACP (Fig 2H) The band intensity indicates that the extent of conversion of refolded holo-ACP is comparable to that of its native counterpart E coli AcpS thus expressed has broad substrate specificity It utilizes apo-ACP and various acyl-CoAs as substrates to give corresponding acyl-ACPs This property of AcpS was utilized to check the extent of the reversibility of folding of apo-ACP An acyl-ACP synthesis assay clearly showed that both native and A B ΔG (kcal/mol) GdnCI concentration [M] 280 290 320 310 300 Temperature [K] m [cal mol–1 M–1] C Fig Effects of temperature on the bestfit Cm (A), m value (B) and DGwater (C) for the guanidine hydrochloride denaturation curves at 10 different temperatures for holoPfACP (d) and apo-PfACP (O) The solid line indicates the best-fit values of Cm (A) and DGwater 330 340 260 320 300 280 Temperature [K] 340 –1 –2 –3 –4 280 FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 290 320 300 310 Temperature [K] 330 340 3319 Plasmodium falciparum acyl carrier protein R Modak et al Discussion Overexpression of several ACPs including E coli ACP, has been reported to be toxic for E coli The active forms of ACP, the holo-ACPs, are even more difficult to overexpress in E coli, presumably due to inefficiency of the E coli holo-ACP synthase in modifying the ACP in vivo, resulting in the production of mostly apo-ACP, which has been shown to have inhibitory effects on E coli growth [26] In our previous studies [24,27] and also in this study, we have standardized conditions for overexpression of PfACP in E coli with high yield (30–35 mgỈL)1 E coli culture) PfACP appears to be converted to holo-ACP by E coli holoACP synthase These studies also show that PfACP is utilized as a substrate by E coli holo-ACP synthase and ACP phosphodiesterase Although optimization of culture conditions yields mostly holo-ACP [27], we show that both holo-ACP and apo-ACP can be overexpressed together and purified to homogeneity The secondary structures of apo-ACP and holoACP, as determined by far-UV CD spectroscopy, have shown the predominance of a-helices and a very low percentage of b-pleated sheet in PfACP Analysis of CD spectra using k2d analysis software (http://www embl-heidelberg.de/$andrade/k2d.html) has shown that both apo-ACP and holo-ACP contain 56% a-helix, 10% b-pleated sheet and 34% random coil in their secondary structure, demonstrating that PfACP has a similar secondary structure to the other ACPs and ACP-like domains [14–22] Hence, detailed biophysical characterization of PfACP could serve as a prototype for determining the conformational stability of other ACPs The NMR structure of PfACP has been solved recently [24,25,27]; this study augments the structural data and elucidates the interactions responsible for the conformational stability of PfACP The size exclusion chromatography profile of PfACP showed that the apparent molecular mass of PfACP monomer is 25 kDa, whereas the actual molecular masses of apo-ACP and holo-ACP are 9.4 kDa and 9.7 kDa, respectively, as is evident from MS studies The dynamic light-scattering experiments showed both apo-ACP and holo-ACP exist as single species [Fig 1Ha,b] The sucrose density gradient sedimentation showed that the apparent molecular masses of apo-ACP and holo-ACP are 16.75 kDa and 21 kDa, respectively The glutaraldehyde crosslinking experiment (Supplementary material) showed that both apoACP and holo-ACP exist as monomers in solution under reducing conditions, and that holo-ACP partially forms a dimer by forming a disulfide bridge involving the SH group of its pantothenyl moiety under 3320 nonreducing conditions only Therefore, the increased apparent molecular masses of monomeric apo-ACP and holo-ACP are not due to oligomerization but are perhaps due to their relatively higher hydrodynamic radii The chaotrope-induced unfolding was almost fully reversible in both forms of the protein Removal of the perturbation makes the protein regain its native form The unfolding reactions of both forms are simple twostate processes, A´U The transitions monitored by the two probes (far-UV CD and tyrosine fluorescence at 305 nm) that report the secondary and tertiary structures of the protein were completely superimposable, thus proving it to be a two-state process [38] Both native and refolded PfACP have comparable mobilities on 12% native PAGE, and both of them are equally utilized as substrates by E coli AcpS and AAS, which further shows the complete refolding of PfACP The fact that it is a small protein with a few hydrophobic residues perhaps explainsd a lack of nonspecific aggregation and the ease with which it can be reversibly unfolded by the chaotrope guanidine hydrochloride Detailed analyses of the stability curves obtained by chemical denaturation are consistent with the LEM The chaotrope-induced equilibrium unfolding of PfACP, followed by fluorescence, fluorescence anisotropy and far-UV CD, showed no evidence for the existence of stable intermediates, substantiating the assumption of a simple two-state transition (Fig 3A,B) Guanidine hydrochloride-induced denaturation experiments on PfACP are consistent with the LEM of protein unfolding [29] It is apparent from the solution denaturation studies that the holo form of the protein has greater stability than the apo form The differences in the unfolding thermodynamic parameters of the two forms are given in Tables 1–3 In the entire experimental regime, it is seen that the holo form presents better stability than the other form (Fig 4C) The DG of stability is on an average 20% greater in the case of the holoprotein as compared to the apoprotein Similarly, the Cm of the holo form always lies above the apo form at all temperatures at which the experiments were conducted The values of Tg, DHg and DCp for the respective Table Thermodynamic parameters of holo-ACP and apo-ACP analyzed on the basis of stability curves drawn for fitting Eqn (6) Protein DHg (kcalỈmol)1) DCp (kcalỈmol)1ỈK)1) Tg (K) Holo-ACP Apo-ACP 53.08 ± 1.09 49.52 ± 1.58 1.18 ± 0.11 1.02 ± 0.13 343.16 ± 1.48 337.20 ± 1.82 FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS R Modak et al proteins were obtained from the fit of Eqn (6) in Experimental procedures It is interesting to note that although the values of DHg and DCp are comparable, the Tg values of the proteins vary slightly; there is a difference of almost °C between the Tg values of the proteins, the value being higher for the holo form There have been contrasting reports about the interaction of the 4¢-PP group with the polypeptide backbone and its effect on the stability of holo-ACP The average major conformation of the holo-ACP NMR structure was analyzed for ligand–protein contacts [30] (http://ligin.weizmann.ac.il/cgi-bin/lpccsu/LpcCsu cgi) to determine the contacts between the 4¢-PP group and polypeptide backbone The greater stability of the holo form may be due to the fact that the 4¢-PP group (structure shown in Fig 5A) makes a number of favorable contacts with the amino acid residues at the surface of the protein by virtue of the presence of several hydrogen bond donors and acceptors in it Furthermore, there are several hydrophobic interactions that hold the structure firmly Closer scrutiny of Fig 5C reveals that whereas most of the surface of the holoACP is lined by charged residues (shown in blue ⁄ red), the interface between the cofactor and the protein is predominantly hydrophobic (represented by gray) Interestingly, a few constructive interactions between carbon and oxygen atoms were also detected at the 4¢-PP–protein interface These favorable interactions might result from atypical CH–O hydrogen bonds According to a report by Jiang et al., these atypical hydrogen bonds play an especially crucial role in stabilizing the protein–protein interface [31] All of the interactions reported in Fig in the range between ˚ and 6.0 A are identical in the major and minor fractions of the holoprotein in solution In fact, the architecture of the bound cofactor in holo-PfACP is like an arch, where the proximal and the distal ends are closer to the protein and the middle portion is away from it Consequently, we notice that although there are a substantial number of attractive interactions between the 4¢-PP group and the protein, they are balanced in a subtle manner This may be because the free movement of the 4¢-PP group is required for its biological activity, and hence extensive contacts between the 4¢-PP group and the peptide backbone may not be a desirable property, which in turn explains the delicate manner in which the stability of holo-ACP is regulated The differences in stability between the two proteins (as indicated by the values of DGwater, Cm and Tg) hence arise from the changes in the surface of the protein because of the interactions of the cofactor with the protein This is further substantiated when one overlays the two forms of the protein (Fig 5B) The Plasmodium falciparum acyl carrier protein ˚ rmsd in this case happens to be 0.20 A Again, differences are seen mostly in the loop regions where the 4¢-PP binds the protein In summary, our studies demonstrate that holo-ACP has higher stability than apo-ACP This work also shows that the 4¢-PP group makes some contacts with the polypeptide that stabilize the holo-ACP structure Experimental procedures Chemicals and reagents Imidazole, kanamycin, dithiothreitol, guanidine hydrochloride, thrombin from bovine plasma, sinapicnic acid, trifluoracetic acid, sucrose and SDS ⁄ PAGE reagents were obtained from Sigma-Aldrich (St Louis, MO) Media components were obtained from Difco (Franklin Lakes, NJ) All other chemicals used were of analytical grade All enzymes were obtained from NEB (Ipswich, MA), MBI Fermentas GmbH (St Leon-Rot, Germany) and Promega (Madison, WI) Strains and plasmids E coli DH5a cells (Gibco BRL, Carlsbad, CA) were used for cloning of the gene pET-28a(+) vector (Novagen, Darmstadt, Germany) and E coli BL21(DE3) cells (Novagen) were used for the expression of PfACP Cloning and expression of PfACP in E coli PfACP was cloned as described previously [27] The plasmid containing PfACP was transformed into E coli BL21(DE3) cells (Novagen) The culture was grown at 37 °C with vigorous shaking (160 r.p.m.) in LB broth (Difco) to a cell density of D600 % The culture was then induced with 0.2 mm isopropyl-b-d-thiogalactopyranoside, and further incubated at 37 °C for h to a D of $2.5 After induction, cells were harvested at 5000 r.p.m for 10 min, and the resultant pellet was stored at ) 70 °C if not used immediately Purification of holo-ACP and apo-ACP All the purification steps were carried out at °C unless otherwise indicated The cell pellet was resuspended in lysis buffer containing 20 mm Tris ⁄ HCl (pH 8.5), 200 mm NaCl, and 10 mm imidazole Lysozyme (2 mg) was added, and the mixture was incubated on ice for 30 Cells were disrupted using a probe-type ultrasonicator (Vibra-Cell; Sonics and Materials, Newtown, CT, USA) MgCl2 and MnCl2 were added to the lysate to final concentrations of 10 and mm, respectively, and the mixture was incubated at 35 °C for h [32] Cell debris was removed by centrifugation at 30 000 g for 30 using a Sorvall RC5C PLUS (Thermo Fisher FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3321 Plasmodium falciparum acyl carrier protein A R Modak et al III II IV I I IV III II B C Fig Interactions of the 4¢-PP moiety with the holo-ACP protein The average major conformation was used for ligand–protein contact analysis (A) The interacting atoms are labeled The green dotted lines indicate hydrophobic interactions, and the blue lines denote CH–O hydrogen bonds The 4¢-PP group is linked to the protein by the Ser37 O-c atom Only residues 31–38 of the protein make extensive contacts with the cofactor For the sake of better understanding of the interactions, the entire figure has been divided into four parts (I, II, III and IV): I, interactions with amino acids 30–33; II, interactions with amino acids 33–34; III, interactions with amino acids 35–37; IV, interactions with ˚ amino acids 37–38 (B) The overlay of the apo (green) and holo (orange) forms of the protein (rmsd ¼ 0.20 A) (C) Diagram showing the nature of the surface in holo-ACP It should be noted that the protein has a greater number of charged exposed surface (indicated by blue ⁄ red) than hydrophobic ones The red line denotes the area in the protein that makes contact with the cofactor Scientific, Waltham, MA, USA) The supernatant obtained was applied to an Ni–nitrilotriacetic acid metal affinity column [agarose resin; (Qiagen, Hildon, Germany)] equili- 3322 brated with the lysis buffer The column was initially washed with column buffer (same as lysis buffer) The protein was eluted using a step gradient of 50 mm to m FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS R Modak et al imidazole, and fractions were tested for purity by 15% SDS ⁄ PAGE The ratio of apo-ACP and holo-ACP was checked by 12% native PAGE Purified PfACP (2 mgỈmL)1) was injected onto a Superdex 75 HR 10 · 300 mm column (Amersham Biosciences, Uppsala, Sweden) equilibrated in 20 mm Tris (pH 8.5) and 200 mm NaCl, connected to an ă AKTA (Amersham Biosciences, Uppsala, Sweden) basic FPLC system to separate the holo-ACP dimer from the mixture of apo-ACP and holo-ACP monomers Purified holo-ACP dimer and the mixture of apo-ACP and holo-ACP monomers were applied to a HI-Trap desalting column (Amersham Biosciences), for buffer exchange to thrombin cleavage buffer (10 mm Na2HPO4, 1.8 mm KH2PO4, 140 mm NaCl, 2.7 mm KCl, 10 mm b -mercaptoethanol, pH 7.3) A thrombin cleavage site was engineered in the pET28a vector immediately after the N-terminal His-tag The N-terminus His-tag of ACP was cleaved by the addition of U of thrombin from bovine plasma (Sigma-Aldrich) per mg of ACP and incubation at 25 °C for h The uncleaved ACP was separated from the cleaved protein by passage through the Ni–nitrilotriacetic acid agarose column The mixture of apo-ACP and holo-ACP monomers was applied to a MonoQ HR ⁄ anion exchange column (Amersham Biosciences) equilibrated with 20 mm Bis ⁄ Tris (pH 6.5) and mm dithiothreitol, and eluted with an NaCl gradient in the same buffer [33] Finally, both apo-ACP and holo-ACP were subjected to buffer exchange in mm Na ⁄ K phosphate (pH 6.5), 100 mm NaCl and mm dithiothreitol using a HI-Trap desalting column, and stored at ) 80 °C until further use PAGE under native conditions Samples were mixed with 6· sample loading buffer (300 mm Tris, pH 6.8, 0.6% bromophenol blue, 60% glycerol) and were analyzed by 12% PAGE without SDS The electrophoresis was performed at room temperature under a constant current of 25 mA per gel The gels were stained with Coomassie Blue Urea PAGE for conformation-sensitive PAGE was prepared similarly, except for the addition of m urea and an increase in the acrylamide concentration to 20% [34] The sample buffer also contained 2.5 m urea Determination of molecular masses of apo-ACP and holo-ACP Purified holo-ACP and apo-ACP were desalted in water using a Hi-Trap desalting column (Amersham Biosciences) Samples were mixed uniformly with lL of the matrix, prepared by adding 0.05% trifluoroacetic acid to a saturated solution of sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid), and spotted onto the MALDI plate The molecular mass was determined with an ULTRA FLEX TOF ⁄ MALDI-TOF mass spectrometer from Bruker Daltonics (Bremen, Germany) Plasmodium falciparum acyl carrier protein Biophysical characterization of ACP CD spectroscopy CD spectra in the far-UV region (200–250 nm) were collected typically at 15 lm protein concentration in a JASCOJ910 polarimeter (JASCO, Tokyo, Japan) in a 0.1 cm pathlength cell, with a slit width of nm, response time of s, and scan speed of 50 nmỈs)1 Isothermal guanidine hydrochloride denaturation studies Denaturant-dependent equilibrium unfolding studies were done by CD and fluorescence spectroscopy Guanidine hydrochloride was prepared in buffer containing mm Na ⁄ K phosphate (pH 6.5), 100 mm NaCl and mm dithiothreitol, and its concentration was determined by refractive index measurements [35] The protein samples were mixed with the desired concentration of the denaturant and incubated at the given temperature for h to reach the chemical equilibrium, and the molar ellipticity at 222 nm was recorded One hour was found to be sufficient to attain equilibrium No aggregation was noted during this time Protein sequence analysis of PfACP showed the presence of a single tyrosine residue but no tryptophan residue in the mature protein Hence, the weak tyrosine fluorescence was used to study equilibrium guanidine hydrochloride-dependent denaturation ACP at 60 lm was used in all the fluorescence studies Fluorescence spectra were recorded on a Jobin-Yvon Horiba fluorometer (Kyoto, Japan) under computer control The excitation and emission monochromator slit widths were and nm, respectively Measurements were performed at 25 °C in a mL quartz cuvette, by exciting the samples at 280 nm and recording the emission between 295 and 350 nm Fluorescence anisotropy experiments The anisotropy experiments for the single tyrosine residue at the C-terminus were performed to study equilibrium guanidine hydrochloride-dependent denaturation A JobinYvon spectrofluorimeter with an excitation slit width of nm and emission of nm was used Samples were excited at 280 nm, and emission was recorded at 305 nm ApoACP and holo-ACP at 60 lm were used for the studies The anisotropy was calculated according to the following equation [36]: Aẳ Iq I? IP ỵ 2I? where I|| is the fluorescence in the parallel direction, and I^ is the fluorescence in the perpendicular direction FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3323 Plasmodium falciparum acyl carrier protein R Modak et al Data analysis According to the linear free energy model [29,37], the changes in the Gibbs free energy, enthalpy, entropy and heat capacity that accompany protein unfolding have a linear dependence on the molar concentration of the denaturant, i.e DG ẳ DGH2O ỵ mẵGdnHCl the change in heat capacity, Cp In order to calculate the change in heat capacity (DCp) for the reaction, we used the method of Pace, where the free energies (DGo) calculated at different temperatures are tted to the GibbsHelmholtz equation DGTị ẳ DHg T=Tg ị ỵ DCp ẵT Tg T lnðT=Tg ފ ð6Þ ð1Þ Acyl-ACP synthesis assay for apo-ACP where DG¢ represents the free energy of unfolding obtained in the presence of a known guanidine hydrochloride concentration The denaturant concentration at which DG ẳ at any temperature is given by the Cm, so that DGH2O ¼ )Cmm [37] For solvent denaturation curves, the key parameter is the m value, defined as the gradient of change in the folding free energy with molar denaturant concentration Isothermal guanidine hydrochloride denaturation curves A simple two-state guanidine hydrochloride-induced denaturation curve determined at a temperature T was analyzed in two different but equivalent ways to obtain DGH2O, the free energy of unfolding in water at temperature T In the first method, a single guanidine hydrochlorideinduced denaturation, where the observed CD signal at each point in the unfolding experiment is mobs, was analyzed with the equation mo ẳ obs m No ỵ aN ẵGdnHCl ỵ D0 ỵ aD ẵGdnHClị expẵRT ẵGdnHCl Cmị m ỵ expẵRT ẵGdnHCl Cmị where No and Do represent the intercept and aN and aD represent the slopes of the folded and unfolded baselines, respectively This expression combines the LEM (Eqn 5), [29] where DGH2O ¼ ) mCm, the two-state assumption for the unfolding reaction, and linear pretransition and posttransition baselines, which are dependent on the concentration of guanidine hydrochloride by the equation Xo + aX [guanidine hydrochloride] In the second method of analysis, the raw data were first converted to plots of fu (fraction unfolded state) vs [guanidine hydrochloride] using the following equations: fu ¼ mo À No ỵ aN ẵGdnHClị obs Do ỵ aD ẵGdnHCl No ỵ aN ẵGdnHClị Keq ẳ ẵU fu fu ẳ ẳ ẵN fn fu DG0 ẳ RTlnKeq 3ị ð4Þ ð5Þ The unfolding of a protein is accompanied by the exposure of the hydrophobic core region, which is reflected in 3324 E coli AcpS [38] was cloned in the pET22b(+) vector as a C-terminal hexa-histidine tag fusion protein in the laboratory The recombinant protein was heterologously expressed in E coli BL21(DE3) cells and purified by Ni–nitrilotriacetic acid affinity chromatography Previously, it was reported that AcpS catalyzes conversion of apo-ACP to holo-ACP through the transfer of the 4¢-PP group from CoA In our studies, we have found that AcpS has broader substrate specificity It utilizes acyl-CoAs of various chain lengths to convert apo-ACP to corresponding acyl-ACPs This property of AcpS was utilized to check the proper refolding of apo-ACP to get a functionally active protein The acyl-ACP synthesis reaction buffer contained mm Na ⁄ K phosphate (pH 6.5), 10 mm MgCl2, 100 mm NaCl and mm dithiothreitol Apo-ACP at 100 lm, AcpS at lm and lauroyl-CoA at 200 lm were used in each assay ð2Þ Both native and refolded apo-ACP were used for the assay The reaction mixtures were incubated at 37 °C for h, and checked by 12% native PAGE The products were confirmed by MS AAS assay for holo-ACP E coli AAS was cloned and expressed in the laboratory as a C-terminal histidine-tagged protein [39] The AAS assay buffer contained 100 mm Tris ⁄ HCl (pH 8), 10 mm MgCl2, 400 mm LiCl, 2% Triton X-100, and mm ATP HoloACP 100 lm, mg of AAS per 100 lL of reaction mixture and 200 lm lauric acid were used per assay The reaction mixtures were incubated at 37 °C for h, and the product was checked by 20% conformation-sensitive PAGE with m urea [36] Dynamic light-scattering studies of PfACP Dynamic light-scattering studies were performed on a Brookhaven Instruments (Holtsville, NY, USA) Dynamic Light Scattering set-up that can measure sizes from to FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS R Modak et al 4000 nm The samples of apo-ACP and holo-ACP in 20 mm Tris (pH 8.0), 200 mm NaCl and mm dithiothreitol were centrifuged at 13 000 g for 15 in a Biofuge centrifuge (Thermo Fisher Scientific, Waltham Mass, MA, USA) and filtered through a 0.1 lm filter The data acquisition time was The routines used to fit the data points were cumulants, and non-negative least-squares analysis was used to obtain the hydrodynamic radii of PfACP A range of PfACP concentrations from mgỈmL)1 (200 lm) to mgỈmL)1 (800 lm) was used for these studies Sucrose density gradient sedimentation Apo-ACP and holo-ACP were layered on top of continuous 0–10% (w ⁄ v) mL sucrose density gradients in a buffer containing 20 mm Tris ⁄ HCl (pH 8), 200 mm NaCl and mm dithiothreitol For sucrose density gradient experiments for holo-ACP under oxidizing conditions, dithiothreitol was omitted The sedimentation was performed with a Beckman SW60Ti rotor at 105 169 g (32 000 r.p.m.) for 20 h at °C Two-hundred-microliter fractions of the gradient were then collected Proteins were analyzed by 12% native PAGE and visualized by silver staining Protein markers, including cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa) and BSA (66 kDa), were separated and fractionated under the same conditions and detected by SDS ⁄ PAGE and silver staining The protein bands were quantified by quantity one (BioRad, Hercules, CA, USA) software Acknowledgements This work was supported by the Department of Biotechnology and the Department of Science and Technology, 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is available online: Doc S1 Crosslinking using glutaraldehyde Fig S1 Oligomeric status of PfACP determined by glutaraldehyde crosslinking This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS ... The chaotrope-induced unfolding was almost fully reversible in both forms of the protein Removal of the perturbation makes the protein regain its native form The unfolding reactions of both forms. .. better stability than the other form (Fig 4C) The DG of stability is on an average 20% greater in the case of the holoprotein as compared to the apoprotein Similarly, the Cm of the holo form always... the interaction of the 4¢-PP group with the polypeptide backbone and its effect on the stability of holo- ACP The average major conformation of the holo- ACP NMR structure was analyzed for ligand–protein