Abstract
Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.
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References
Ahern TJ, Klibanov AM (1985) The mechanisms of irreversible enzyme inactivation at 100°C. Science 228:1280–1284. https://doi.org/10.1126/science.4001942
Almén MS, Nordström KJV, Fredriksson R, Schiöth HB (2009) Map** the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin. BMC Biol 7:50. https://doi.org/10.1186/1741-7007-7-50
Andersen KK, Wang H, Otzen DE (2012) A kinetic analysis of the folding and unfolding of OmpA in urea and guanidinium chloride: single and parallel pathways. Biochemistry 51:8371–8383. https://doi.org/10.1021/bi300974y
Barrera FN, Renart ML, Molina ML, Poveda JA, Encinar JA, Fernández AM, Neira JL, González-Ros JM (2005) Unfolding and refolding in vitro of a tetrameric, α-helical membrane protein: the prokaryotic potassium channel KcsA. Biochemistry 44:14344–14352. https://doi.org/10.1021/bi050845t
Booth PJ, Curnow P (2009) Folding scene investigation: membrane proteins. Curr Opin Struct Biol 19:8–13. https://doi.org/10.1016/j.sbi.2008.12.005
Broom A, Ma SM, **a K, Rafalia H, Trainor K, Colón W, Gosavi S, Meiering EM (2015) Designed protein reveals structural determinants of extreme kinetic stability. Proc Natl Acad Sci U S A 112:14605–14610. https://doi.org/10.1073/pnas.1510748112
Buchanan SK, Yamashita S, Fleming KG (2012) Structure and folding of outer membrane proteins. Comp Biophys 5:139–163. https://doi.org/10.1016/B978-0-12-374920-8.00514-2
Burgess NK, Dao TP, Stanley AM, Fleming KG (2008) β-Barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro. J Biol Chem 283:26748–26758. https://doi.org/10.1074/jbc.M802754200
Cattoni DI, González Flecha FL, Argüello JM (2008) Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus. Arch Biochem Biophys 471:198–206. https://doi.org/10.1016/j.abb.2007.12.013
Cattoni DI, Kaufman SB, González Flecha FL (2009) Kinetics and thermodynamics of the interaction of 1-anilino-naphthalene-8-sulfonate with proteins. Biochim Biophys Acta 1794:1700–1708. https://doi.org/10.1016/j.bbapap.2009.08.007
Corley SC, Sprangers P, Albert AD (2011) The bilayer enhances rhodopsin kinetic stability in bovine rod outer segment disk membranes. Biophys J 100:2946–2954. https://doi.org/10.1016/j.bpj.2011.05.015
Creighton TE (1996) Proteins: structures and molecular properties, 2nd edn. Freeman, New York
Curnow P, Booth PJ (2007) Combined kinetic and thermodynamic analysis of α-helical membrane protein unfolding. Proc Nat Acad Sci U S A 104:18970–18975. https://doi.org/10.1073/pnas.0705067104
Curnow P, Booth PJ (2009) The transition state for integral membrane protein folding. Proc Natl Acad Sci U S A 106:773–778. https://doi.org/10.1073/pnas.0806953106
Danoff EJ, Fleming KG (2017) Novel kinetic intermediates populated along the folding pathway of the transmembrane β-barrel OmpA. Biochemistry 56:47–60. https://doi.org/10.1021/acs.biochem.6b00809
Di Bartolo N, Compton ELR, Warne T, Edwards PC, Tate CG, Schertler GFX, Booth PJ (2016) Complete reversible refolding of a G-protein coupled receptor on a solid support. PLoS One 11:e0151582. https://doi.org/10.1371/journal.pone.0151582
Dodes Traian MM, Cattoni DI, Levi V, González Flecha FL (2012) A two-stage model for lipid modulation of the activity of integral membrane proteins. PLoS One 7:e39255. https://doi.org/10.1371/journal.pone.0039255
Faham S, Yang D, Bare E, Yohannan S, Whitelegge JP, Bowie JU (2004) Side-chain contributions to membrane protein structure and stability. J Mol Biol 335:297–305. https://doi.org/10.1016/j.jmb.2003.10.041
Fleming KG (2014) Energetics of membrane protein folding. Annu Rev Biophys 43:233–255. https://doi.org/10.1146/annurev-biophys-051013-022926
Galisteo ML, Sanchez-Ruiz JM (1993) Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin. Eur Biophys J 22:25–30. https://doi.org/10.1007/bf00205809
González-Lebrero RM, Kaufman SB, Garrahan PJ, Rossi RC (2002) The occlusion of Rb(+) in the Na(+)/K(+)-ATPase. II. The effects of Rb(+), Na(+), Mg2(+), or ATP on the equilibrium between free and occluded Rb(+). J Biol Chem 277:5922–5928. https://doi.org/10.1074/jbc.M105887200
Greene RF, Pace CN (1974) Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, α-chymotrypsin, and β-lactoglobulin. J Biol Chem 249:5388–5393
Gursky O (2015) Structural stability and functional remodeling of high-density lipoproteins. FEBS Lett 589:2627–2639. https://doi.org/10.1016/j.febslet.2015.02.028
Hänggi P, Talkner P, Borkovec M (1990) Reaction-rate theory: fifty years after Kramers. Rev Mod Phys 62:251–341. https://doi.org/10.1103/RevModPhys.62.251
Harris NJ, Booth PJ (2012) Folding and stability of membrane transport proteins in vitro. Biochim Biophys Acta 1818:1055–1066. https://doi.org/10.1016/j.bbamem.2011.11.006
Harris NJ, Findlay HE, Sanders MR, Kedzierski M, dos Santos Á, Booth PJ (2017) Comparative stability of major facilitator superfamily transport proteins. Eur Biophys J 46:655–663. https://doi.org/10.1007/s00249-017-1197-7
Hong H, Tamm LK (2004) Elastic coupling of integral membrane protein stability to lipid bilayer forces. Proc Natl Acad Sci U S A 101:4065–4070. https://doi.org/10.1073/pnas.0400358101
Hong H, Joh NH, Bowie JU, Tamm LK (2009) Methods for measuring the thermodynamic stability of membrane proteins. Methods Enzymol 455:213–236. https://doi.org/10.1016/S0076-6879(08)04208-0
Huysmans GHM, Baldwin SA, Brockwell DJ, Radford SE (2010) The transition state for folding of an outer membrane protein. Proc Natl Acad Sci U S A 107:4099–4104. https://doi.org/10.1073/pnas.0911904107
Jefferson RE, Blois TM, Bowie JU (2013) Membrane proteins can have high kinetic stability. J Am Chem Soc 135:15183–15190. https://doi.org/10.1021/ja407232b
Jefferson RE, Min D, Corin K, Wang JY, Bowie JU (2017) Applications of single-molecule methods to membrane protein folding studies. J Mol Biol 429. https://doi.org/10.1016/j.jmb.2017.05.021
Karshikoff A, Nilsson L, Ladenstein R (2015) Rigidity versus flexibility: the dilemma of understanding protein thermal stability. FEBS J 282:3899–3917. https://doi.org/10.1111/febs.13343
Kaufman SB, González-Flecha FL, González-Lebrero RM (2012) Opposing effects of Na+ and K+ on the thermal stability of Na+,K+-ATPase. J Phys Chem B 116:3421–3429. https://doi.org/10.1021/jp2124108
Kemmer G, Keller S (2010) Nonlinear least-squares data fitting in Excel spreadsheets. Nat Protocols 5:267–281. https://doi.org/10.1038/nprot.2009.182
Kleinschmidt JH (2015) Folding of β-barrel membrane proteins in lipid bilayers—unassisted and assisted folding and insertion. Biochim Biophys Acta 1848:1927–1943. https://doi.org/10.1016/j.bbamem.2015.05.004
Kramers HA (1940) Brownian motion in a field of force and the diffusion model of chemical reactions. Physica 7:284–304. https://doi.org/10.1016/S0031-8914(40)90098-2
Laidler KJ, King MC (1983) Development of transition-state theory. J Phys Chem 87:2657–2664. https://doi.org/10.1021/j100238a002
Lau FW, Bowie JU (1997) A method for assessing the stability of a membrane protein. Biochemistry 36:5884–5892. https://doi.org/10.1021/bi963095j
Levi V, Rossi JPFC, Echarte MM, Castello PR, González Flecha FL (2000) Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid–protein interactions. J Memb Biol 173:215–225. https://doi.org/10.1007/s002320001021
Levi V, Rossi JPFC, Castello PR, González Flecha FL (2002) Structural significance of the plasma membrane calcium pump oligomerization. Biophys J 82:437–446. https://doi.org/10.1016/S0006-3495(02)75408-8
Levi V, Rossi JP, Castello PR, González Flecha FL (2003) Quantitative analysis of membrane protein–amphiphile interactions using resonance energy transfer. Anal Biochem 317:171–179. https://doi.org/10.1016/S0003-2697(03)00132-5
Levi V, Villamil Giraldo AM, Castello PR, Rossi JPFC, González Flecha FL (2008) Effects of phosphatidylethanolamine glycation on lipid–protein interactions and membrane protein thermal stability. Biochem J 416:145–152. https://doi.org/10.1042/BJ20080618
Lumry R, Eyring H (1954) Conformation changes of proteins. J Phys Chem 58:110–120. https://doi.org/10.1021/j150512a005
MacKenzie KR (2006) Folding and stability of α-helical integral membrane proteins. Chem Rev 106:1931–1977. https://doi.org/10.1021/cr0404388
Manning M, Colón W (2004) Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward β-sheet structure. Biochemistry 43:11248–11254. https://doi.org/10.1021/bi0491898
Marsh D (2008) Protein modulation of lipids, and vice-versa, in membranes. Biochim Biophys Acta 1778:1545–1575. https://doi.org/10.1016/j.bbamem.2008.01.015
Moon CP, Fleming KG (2011a) Side-chain hydrophobicity scale derived from transmembrane protein folding into lipid bilayers. Proc Natl Acad Sci U S A 108:10174–10177. https://doi.org/10.1073/pnas.1103979108
Moon CP, Fleming KG (2011b) Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes. Methods Enzymol 492:189–211. https://doi.org/10.1016/B978-0-12-381268-1.00018-5
Myers JK, Pace CN, Scholtz JM (1995) Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci 4:2138–2148. https://doi.org/10.1002/pro.5560041020
Nury S, Meunier JC (1990) Molecular mechanisms of the irreversible thermal denaturation of guinea-pig liver transglutaminase. Biochem J 266:487–490. https://doi.org/10.1042/bj2660487
Otzen DE (2003) Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein. J Mol Biol 330:641–649. https://doi.org/10.1016/S0022-2836(03)00624-7
Otzen DE, Andersen KK (2013) Folding of outer membrane proteins. Arch Biochem Biophys 531:34–43. https://doi.org/10.1016/j.abb.2012.10.008
Panigrahi R, Arutyunova E, Panwar P, Gimpl K, Keller S, Lemieux MJ (2016) Reversible unfolding of rhomboid intramembrane proteases. Biophys J 110:1379–1390. https://doi.org/10.1016/j.bpj.2016.01.032
Park K, Perczel A, Fasman GD (1992) Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins. Protein Sci 1:1032–1049. https://doi.org/10.1002/pro.5560010809
Park C, Zhou S, Gilmore J, Marqusee S (2007) Energetics-based protein profiling on a proteomic scale: identification of proteins resistant to proteolysis. J Mol Biol 368:1426–1437. https://doi.org/10.1016/j.jmb.2007.02.091
Placenti MA, Kaufman SB, González Flecha FL, González Lebrero RM (2017) Unexpected effects of K+ and adenosine triphosphate on the thermal stability of Na+,K+-ATPase. J Phys Chem B 121:4949–4957. https://doi.org/10.1021/acs.jpcb.7b00629
Pocanschi CL, Popot J-L, Kleinschmidt JH (2013) Folding and stability of outer membrane protein a (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35. Eur Biophys J 42:103–118. https://doi.org/10.1007/s00249-013-0887-z
Popot JL, Engelman DM (1990) Membrane protein folding and oligomerization: the two-stage model. Biochemistry 29:4031–4037. https://doi.org/10.1021/bi00469a001
Popot JL, Engelman DM (2000) Helical membrane protein folding, stability, and evolution. Annu Rev Biochem 69:881–922. https://doi.org/10.1146/annurev.biochem.69.1.881
Powl AM, Miles AJ, Wallace BA (2012) Transmembrane and extramembrane contributions to membrane protein thermal stability: studies with the NaChBac sodium channel. Biochim Biophys Acta 1818:889–895. https://doi.org/10.1016/j.bbamem.2011.12.019
Privalov PL (1979) Stability of proteins: small globular proteins. Adv Prot Chem 33:167–241. https://doi.org/10.1016/S0065-3233(08)60460-X
Pucci F, Rooman M (2017) Physical and molecular bases of protein thermal stability and cold adaptation. Curr Op Struct Biol 42:117–128. https://doi.org/10.1016/j.sbi.2016.12.007
Quezada AG, Díaz-Salazar AJ, Cabrera N, Pérez-Montfort R, Piñeiro Á, Costas M (2017) Interplay between protein thermal flexibility and kinetic stability. Structure 25:167–179. https://doi.org/10.1016/j.str.2016.11.018
Roman EA, González Flecha FL (2014) Kinetics and thermodynamics of membrane protein folding. Biomol Ther 4:354–373. https://doi.org/10.3390/biom4010354
Roman EA, Argüello JM, González Flecha FL (2010) Reversible unfolding of a thermophilic membrane protein in phospholipid/detergent mixed micelles. J Mol Biol 397:550–559. https://doi.org/10.1016/j.jmb.2010.01.045
Romero-Romero ML, Risso VA, Martinez-Rodriguez S, Gaucher EA, Ibarra-Molero B, Sanchez-Ruiz JM (2016) Selection for protein kinetic stability connects denaturation temperatures to organismal temperatures and provides clues to archaean life. PLoS One 11:e0156657. https://doi.org/10.1371/journal.pone.0156657
Roman EA, Santos J, González Flecha FL (2011) The use of circular dichroism methods to monitor unfolding transitions in peptides, globular and membrane proteins. In: Rodgers DS (ed) Circular dichroism: theory and spectroscopy. Nova Publishers, New York, pp 217–254
Sanchez-Ruiz JM (1992) Theoretical analysis of Lumry–Eyring models in differential scanning calorimetry. Biophys J 61:921–935. https://doi.org/10.1016/S0006-3495(92)81899-4
Sanchez-Ruiz JM (2010) Protein kinetic stability. Biophys Chem 148:1–15. https://doi.org/10.1016/j.bpc.2010.02.004
Schellman JA (1987) The thermodynamic stability of proteins. Annu Rev Biophys 16:115–137. https://doi.org/10.1146/annurev.bb.16.060187.000555
Schellman JA (2002) Fifty years of solvent denaturation. Biophys Chem 96:91–101. https://doi.org/10.1016/S0301-4622(02)00009-1
Schlebach JP, Kim M-S, Joh NH, Bowie JU, Park C (2011) Probing membrane protein unfolding with pulse proteolysis. J Mol Biol 406:545–551. https://doi.org/10.1016/j.jmb.2010.12.018
Schlebach JP, Woodall NB, Bowie JU, Park C (2014) Bacteriorhodopsin folds through a poorly organized transition state. J Am Chem Soc 136:16574–16581. https://doi.org/10.1021/ja508359n
Sedlák E, Varhač R, Musatov A, Robinson NC (2014) The kinetic stability of cytochrome c oxidase: effect of bound phospholipid and dimerization. Biophys J 107:2941–2949. https://doi.org/10.1016/j.bpj.2014.10.055
Shortle D, Ackerman MS (2001) Persistence of native-like topology in a denatured protein in 8 M urea. Science 293:487–489
Sreerama N, Venyaminov SY, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 287:243–251. https://doi.org/10.1006/abio.2000.4879
Tanford C (1968) Protein denaturation. Adv Prot Chem 23:121–282. https://doi.org/10.1016/S0065-3233(08)60401-5
Tanford C (1980) The hydrophobic effect: formation of micelles and biological membranes. Wiley-Interscience, New York
Torrent J, Marchal S, Ribó M, Vilanova M, Georges C, Dupont Y, Lange R (2008) Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps. Biophys J 94:4056–4065. https://doi.org/10.1529/biophysj.107.123893
von Heijne G (1986) The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J 5:3021–3027
Wallin E, Von Heijne G (1998) Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Prot Sci 7:1029–1038. https://doi.org/10.1002/pro.5560070420
Wang W, Roberts CJ (2013) Non-Arrhenius protein aggregation. AAPS J 15:840–851. https://doi.org/10.1208/s12248-013-9485-3
White SH, Wimley WC (1999) Membrane protein folding and stability: physical principles. Annu Rev Biophys Biomol Struct 28:319–365. https://doi.org/10.1146/annurev.biophys.28.1.319
Yan YB, Wang Q, He HW, Zhou HM (2004) Protein thermal aggregation involves distinct regions: sequential events in the heat-induced unfolding and aggregation of hemoglobin. Biophys J 86:1682–1690. https://doi.org/10.1016/S0006-3495(04)74237-X
Yildirim MA, Goh K-I, Cusick ME, Barabási A-L, Vidal M (2007) Drug–target network. Nat Biotech 25:1119–1126. https://doi.org/10.1038/nbt1338
Yoneda JS, Rigos CF, Ciancaglini P (2013) Addition of subunit γ, K+ ions, and lipid restores the thermal stability of solubilized Na,K-ATPase. Arch Biochem Biophys 530:93–100. https://doi.org/10.1016/j.abb.2012.12.022
Yoneda JS, Scanavachi G, Sebinelli HG, Borges JC, Barbosa LRS, Ciancaglini P, Itri R (2016) Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase. Int J Biol Macromol 89:238–245. https://doi.org/10.1016/j.ijbiomac.2016.04.058
Zhou Y, Lau FW, Nauli S, Yang D, Bowie JU (2001) Inactivation mechanism of the membrane protein diacylglycerol kinase in detergent solution. Protein Sci 10:378–383. https://doi.org/10.1110/ps.34201
Zhu Y, Alonso DO, Maki K, Huang CY, Lahr SJ, Daggett V, Roder H, DeGrado WF, Gai F (2003) Ultrafast folding of α3D: a de novo designed three-helix bundle protein. Proc Natl Acad Sci U S A 100:15486–15491. https://doi.org/10.1073/pnas.2136623100
Acknowledgements
The author thanks Dr. Rodolfo M González-Lebrero for the helpful discussions. This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT2013-1691), CONICET (PIP 2013-00014CO), and Universidad de Buenos Aires (UBACyT 20020130100460BA).
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This article is part of a Special Issue on ‘Latin America’ edited by Pietro Ciancaglini and Rosangela Itri.
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González Flecha, F.L. Kinetic stability of membrane proteins. Biophys Rev 9, 563–572 (2017). https://doi.org/10.1007/s12551-017-0324-0
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DOI: https://doi.org/10.1007/s12551-017-0324-0