Abstract
Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has α-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.
Similar content being viewed by others
Abbreviations
- ANS:
-
8-Anilino-1-naphthalenesulfonic acid
- BSA:
-
Bovine serum albumin
- CD:
-
Circular dichorism
- DSC:
-
Differential scanning calorimetry
- FPLC:
-
Fast protein liquid chromatography
- GuHCl:
-
Guanidine hydrochloride
- GuSCN:
-
Guanidine isothiocynate
- UV:
-
Ultraviolet
References
Barry JK, Matthews KS (1999) Thermodynamic analysis of unfolding and dissociation in lactose repressor protein. Biochemistry 38:6520–6528
Chen P, Tsuge H, Almassy RJ, Gribskov CL, Katoh S, Vanderpool DL, Margosiak SA, Pinko C, Matthews DA, Kan CC (1996) Structure of the human cytomegalovirus protease catalytic domain reveals a novel serine protease fold and catalytic triad. Cell 86:835–843
Clark AC, Sinclair JF, Baldwin TO (1993) Folding of bacterial luciferase involves a non-native heterodimeric intermediate in equilibrium with the native enzyme and the unfolded subunits. J Biol Chem 268:10773–10779
Cunningham EL, Jaswal SS, Sohl JL, Agard DA (1999) Kinetic stability as a mechanism for protease longevity. Proc Natl Acad Sci U S A 96:11008–11014
Dams T, Jaenicke R (1999) Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima. Biochemistry 38:9169–9178
Dong A, Lam T (2005) Equilibrium titrations of acid-induced unfolding-refolding and salt-induced molten globule of cytochrome c by FT-IR spectroscopy. Arch Biochem Biophys 436:154–160
Edwin F, Jagannadham MV (1998) Sequential unfolding of papain in molten globule state. Biochem Biophys Res Commun 252:654–660
Edwin F, Jagannadham MV (2000) Single disulfide bond reduced papain exists in a compact intermediate state. Biochim Biophys Acta 1479:69–82
Edwin F, Sharma YV, Jagannadham MV (2002) Stabilization of molten globule state of papain by urea. Biochem Biophys Res Commun 290:1441–1446
Fink AL (1995) Compact intermediate states in protein folding. Annu Rev Biophys Biomol Struct 24:495–522
Gittelman MS, Matthews CR (1990) Folding and stability of trp aporepressor from Escherichia coli. Biochemistry 29:7011–7020
Go N (1983) Theoretical studies of protein folding. Annu Rev Biophys Bioeng 12:183–210
Guidry JJ, Moczygemba CK, Steede NK, Landry SJ, Wittung-Stafshede P (2000) Reversible denaturation of oligomeric human chaperonin 10: denatured state depends on chemical denaturant. Protein Sci 9:2109–2117
Herold M, Kirschner K (1990) Reversible dissociation and unfolding of aspartate aminotransferase from Escherichia coli: characterization of a monomeric intermediate. Biochemistry 29:1907–1913
Hink-Schauer C, Estebanez-Perpina E, Kurschus FC, Bode W, Jenne DE (2003) Crystal structure of the apoptosis-inducing human granzyme A dimer. Nat Struct Biol 10:535–540
Hornby JA, Luo JK, Stevens JM, Wallace LA, Kaplan W, Armstrong RN, Dirr HW (2000) Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate. Biochemistry 39:12336–12344
Jaswal SS, Sohl JL, Davis JH, Agard DA (2002) Energetic landscape of alpha-lytic protease optimizes longevity through kinetic stability. Nature 415:343–346
Jones MN, Skinner HA, Tipping E (1975) The interaction between bovine serum albumin and surfactants. Biochem J 147:229–234
Kim PS, Baldwin RL (1982) Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu Rev Biochem 51:459–489
Kim YR, Hahn JS, Hong H, Jeong W, Song NW, Shin HC, Kim D (1999) Dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha induced by guanidine hydrochloride. Biochim Biophys Acta 1429:486–495
Kuwajima K (1989) The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins 6:87–103
Manavalan P, Johnson WC Jr (1983) Sensitivity of circular dichroism to protein tertiary structure class. Nature 305:831
Manavalan P, Johnson WC Jr, Modrich P (1984) Prediction of secondary structure for Eco RI endonuclease. J Biol Chem 259:11666–11667
Manning M, Colon W (2004) Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward beta-sheet structure. Biochemistry 43:11248–11254
Mei G, Di Venere A, Rosato N, Finazzi-Agro A (2005) The importance of being dimeric. Febs J 272:16–27
Milla ME, Sauer RT (1994) P22 Arc repressor: folding kinetics of a single-domain, dimeric protein. Biochemistry 33:1125–1133
Pace CN (1990) Measuring and increasing protein stability. Trends Biotechnol 8:93–98
Panse VG, Swaminathan CP, Aloor JJ, Surolia A, Varadarajan R (2000) Unfolding thermodynamics of the tetrameric chaperone, SecB. Biochemistry 39:2362–2369
Pettit SC, Gulnik S, Everitt L, Kaplan AH (2003) The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage. J Virol 77:366–374
Ramstein J, Hervouet N, Coste F, Zelwer C, Oberto J, Castaing B (2003) Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure. J Mol Biol 331:101–121
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635
Reddy GB, Bharadwaj S, Surolia A (1999) Thermal stability and mode of oligomerization of the tetrameric peanut agglutinin: a differential scanning calorimetry study. Biochemistry 38:4464–4470
Redfield RR, Anfinsen CB (1956) The structure of ribonuclease. II. The preparation, separation, and relative alignment of large enzymatically produced fragments. J Biol Chem 221:385–404
Reynolds JA, Herbert S, Polet H, Steinhardt J (1967) The binding of divers detergent anions to bovine serum albumin. Biochemistry 6:937–947
Risse B, Stempfer G, Rudolph R, Mollering H, Jaenicke R (1992) Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction. Protein Sci 1:1699–1709
Schiffer CA, Dotsch V (1996) The role of protein–solvent interactions in protein unfolding. Curr Opin Biotechnol 7:428–432
Service RF (2008) Problem solved* (*sort of). Science 321:784–786
Sharma YV, Jagannadham MV (2003) N-terminal domain unfolds first in the sequential unfolding of papain. Protein Pept Lett 10:83–90
Silinski P, Fitzgerald MC (2002) A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT). Biochemistry 41:4480–4491
Smith CJ, Clarke AR, Chia WN, Irons LI, Atkinson T, Holbrook JJ (1991) Detection and characterization of intermediates in the folding of large proteins by the use of genetically inserted tryptophan probes. Biochemistry 30:1028–1036
Spelbrink RE, Kolkman A, Slijper M, Killian JA, de Kruijff B (2005) Detection and identification of stable oligomeric protein complexes in Escherichia coli inner membranes: a proteomics approach. J Biol Chem 280:28742–28748
Sundd M, Kundu S, Jagannadham MV (2002) Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates. J Biochem Mol Biol 35:143–154
Watt SJ, Sheil MM, Beck JL, Prosselkov P, Otting G, Dixon NE (2007) Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra. J Am Soc Mass Spectrom 18:1605–1611
Xia K, Manning M, Hesham H, Lin Q, Bystroff C, Colon W (2007) Identifying the subproteome of kinetically stable proteins via diagonal 2D SDS/PAGE. Proc Natl Acad Sci U S A 104:17329–17334
Yadav SC, Jagannadham MV (2008) Physiological changes and molluscicidal effects of crude latex and Milin on Biomphalaria glabrata. Chemosphere 71:1295–1300
Yadav SC, Jagannadham MV (2009) Complete conformational stability of kinetically stable dimeric serine protease milin against pH, temperature, urea, and proteolysis. Eur Biophys J 38:981–991
Yadav SC, Pande M, Jagannadham MV (2006) Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii. Phytochemistry 67:1414–1426
Yadav SC, Jagannadham MV, Kundu S, Jagannadham MV (2009) A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties. Biophys Chem 139:13–23
Yuan C, Li J, Selby TL, Byeon IJ, Tsai MD (1999) Tumor suppressor INK4: comparisons of conformational properties between p16(INK4A) and p18(INK4C). J Mol Biol 294:201–211
Zaks A, Klibanov AM (1988) Enzymatic catalysis in nonaqueous solvents. J Biol Chem 263:3194–3201
Zhuang P, Eisenstein E, Howell EE (1994) Equilibrium folding studies of tetrameric R67 dihydrofolate reductase. Biochemistry 33:4237–4244
Ziegler MM, Goldberg ME, Chaffotte AF, Baldwin TO (1993) Refolding of luciferase subunits from urea and assembly of the active heterodimer. Evidence for folding intermediates that precede and follow the dimerization step on the pathway to the active form of the enzyme. J Biol Chem 268:10760–10765
Acknowledgments
The financial assistance to SCY from CSIR, Government of India, in the form of a research fellowship and to MBU, IMS, BHU from UGC and DBT, Government of India, for infrastructure is acknowledged. Appreciation is also extended to the University of Delhi, New Delhi, and DBT for financial and infrastructural assistance to SK. We are thankful to Prof. Rajiv Bhat, JNU, New Delhi, for providing the DSC facility. We are especially thankful to Prof. Vinod Bhakuni, CDRI, Lucknow, for his help in the CD data verification.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Yadav, S.C., Jagannadham, M.V. & Kundu, S. Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates. Eur Biophys J 39, 1385–1396 (2010). https://doi.org/10.1007/s00249-010-0593-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-010-0593-z