Osmolyte-Induced Folding and Stability of Proteins: Concepts and Characterization

Document Type : Review Paper


1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

2 Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.


It is well-known that the typical protein’s three-dimensional structure is relatively unstable in harsh conditions. A practical approach to maintain the folded state and thus improve the stability and activity of proteins in unusual circumstances is to directly apply stabilizing substances such as osmolytes to the protein-containing solutions. Osmolytes as natural occurring organic molecules typically called “compatible” solutes, based on the concept that they do not perturb cellular components. However, urea and guanidine hydrochloride (GuHCl) as denaturing osmolytes destabilize many macromolecular structures and inhibit functions. Several studies have been so far performed to explain the actual interaction of an osmolyte with a protein. The present review is aimed to achieve a collective knowledge of the progress arise in the field of osmolyte-protein interactions. The following is also an overview of the main techniques to measure protein stability in the presence of osmolytes.


Main Subjects

(1) Harano Y and Masahiro K. On the physics of pressure
denaturation of proteins. J. Phys. Condens. Matter.
(2006) 7: L107.
(2) Yoshioka S, Kelly MF, Yukio A and Michael JP. Effect
of sugars on the molecular motion of freeze-dried
protein formulations reflected by NMR relaxation
times. Pharm. Res. (2011) 12: 3237–47.
(3) Neurath H, Greenstein JP, Putnam FW and Erickson
JA. The chemistry of protein denaturation. Chem.
Rev. (1944) 5: 157–265.
(4) Gruebele M. The fast protein folding problem. Annu.
Rev. Phys. Chem. (1999) 1: 485–516.
(5) Pande VS, Grosberg AY and Tanaka T. Heteropolymer
freezing and design: towards physical models of
protein folding. Rev. Mod. Phys. (2000) 1: 259–86.
(6) Adrangi S and Faramarzi MA. From bacteria to
human: a journey into the world of chitinases.
Biotechnol. Adv. (2013) 8: 1786–95.
(7) Kuzmenkina EV, Heyes CD and Nienhaus GU.
Single-molecule Förster resonance energy transfer
study of protein dynamics under denaturing
conditions. Proc. Natl. Acad. Sci., India (2005) 43:
(8) Onuchic JN, Luthey-Schulten Z and Wolynes PG.
Theory of protein folding: the energy landscape
perspective. Annu. Rev. Phys. Chem. (1997) 1: 545–
(9) Harvey SR, Seffernick JT, Quintyn RS, Song Y, Ju
Y, Yan J, Sahasrabuddhe AN, Norris A, Zhou M,
Behrman EJ and Lindert S. Relative interfacial
cleavage energetics of protein complexes revealed
by surface collisions. Proc. Natl. Acad. Sci. (2019)
17: 8143–8.
(10) Poddar NK, Ansari ZA, Singh RB, MoosaviMovahedi AA and Ahmad F. Effect of monomeric
and oligomeric sugar osmolytes on ΔGD, the Gibbs
energy of stabilization of the protein at different
pH values: Is the sum effect of monosaccharide
individually additive in a mixture. Biophys. Chem.
(2008) 3: 120–9.
(11) Han Y, Jin BS, Lee SB, Sohn Y, Joung JW and Lee
JH. Effects of sugar additives on protein stability
of recombinant human serum albumin during
lyophilization and storage. Arch. Pharmacal. Res.
(2007) 9: 1124.
(12) Xie G and Timasheff SN. Mechanism of the
stabilization of ribonuclease A by sorbitol:
preferential hydration is greater for the denatured
than for the native protein. Protein Sci. (1997) 1:
(13) Wang A and Bolen DW. Effect of proline on lactate
dehydrogenase activity: testing the generality and
scope of the compatibility paradigm. Biophys. J.
(1996) 71: 2117–22.
(14) Haque I, Singh R, Moosavi-Movahedi AA and
Ahmad F. Effect of polyol osmolytes on ΔGD, the 
Osmolyte-Induced Folding and Stability of Proteins
Gibbs energy of stabilization of proteins at different
pH values. Biophys. Chem. (2005) 117: 1–2.
(15) Xie G and Timasheff SN. Temperature dependence
of the preferential interactions of ribonuclease a
in aqueous co-solvent systems: Thermodynamic
analysis. Protein Sci. (1997) 1: 222–32.
(16) Welch WJ and Brown CR. Influence of molecular
and chemical chaperones on protein folding. Cell
Stress Chaperones (1996) 2: 109.
(17) Manning MC, Chou DK, Murphy BM, Payne
RW and Katayama DS. Stability of protein
pharmaceuticals: an update. Pharm. Res. (2010) 4:
(18) Robinson NE and Robinson AB. Prediction of
primary structure deamidation rates of asparaginyl
and glutaminyl peptides through steric and catalytic
effects. Int. J. Pept. Res. (2004) 5: 437–48.
(19) Chelius D, Rehder DS and Bondarenko PV.
Identification and characterization of deamidation
sites in the conserved regions of human
immunoglobulin gamma antibodies. Anal Chem.
(2005) 77: 6004–11.
(20) Kossiakoff AA. Tertiary structure is a principal
determinant to protein deamidation. Science (1988)
240: 191–4.
(21) Harris RJ, Shire SJ and Winter C. Commercial
manufacturing scale formulation and analytical
characterization of therapeutic recombinant
antibodies. Drug Dev. Res. (2004) 61: 137–54.
(22) Oliyai C and Borchardt RT. Chemical pathways
of peptide degradation. VI. Effect of the primary
sequence on the pathways of degradation of
aspartyl residues in model hexapeptides. Pharm.
Res. (1994) 11:751–8.
(23) Fisher P. Diketopiperazines in peptide and
combinatorial chemistry. J. Pept. Sci. (2003) 9:
(24) Marsden BJ, Nguyen TMD and Schiller PW.
Spontaneous degradation via diketopiperazine
formation of peptides containing a
tetrahydroisoquinoline-3-carboxylic acid residue
in the 2-position of the peptide sequence. Int. J.
Pept. Protein Res. (1993) 41: 313–6.
(25) Goolcharran C, Khossravi M and Borchardt
RT. Chemical pathways of peptide and protein
degradation. In: Frokjaer S and Hovgaard L.
(eds.). Pharmaceutical formulation development
of peptides and proteins. 2nd ed. CRC Press, New
York (2000) 70–88.
(26) Payne RW and Manning MC. Peptide formulation:
challenges and strategies. Innovations Pharm.
Technol. (2009) 28: 64–8.
(27) Ferreira LA, Uversky VN and Zaslavsky BY. Role
of solvent properties of water in crowding effects
induced by macromolecular agents and osmolytes.
Mol. BioSyst. (2017) 12: 2551–63.
(28) Khan SH, Ahmad N, Ahmad F and Kumar R.
Naturally occurring organic osmolytes: from cell
physiology to disease prevention. IUBMB Life
(2010) 62: 891–5.
(29) P Melo E, Estrela N, Lopes C, C Matias A, Tavares
E and Ochoa-Mendes V. Compacting proteins:
pros and cons of osmolyte-induced folding. Curr.
Protein Pept. Sci. (2010) 11: 744–51.
(30) Singh SK, Kolhe P, Mehta AP, Chico SC, Lary AL
and Huang M. Frozen state storage instability of a
monoclonal antibody: aggregation as a consequence
of trehalose crystallization and protein unfolding.
Pharm. Res. (2011) 4: 873–85.
(31) Usha R and Ramasami T. Stability of collagen with
polyols against guanidine denaturation. Colloids
Surf. B (2008) 1: 39–42.
(32) Macchi F, Eisenkolb M, Kiefer H and Otzen DE.
The effect of osmolytes on protein fibrillation. Int.
J. Mol. Sci. (2012) 3: 3801–3819.
(33) Saunders AJ, Davis-Searles PR, Allen DL, Pielak
GJ and Erie DA. Osmolyte-induced changes in
protein conformational equilibria. Biopolymers
(2000) 4: 293–307.
(34) Mondal J., Halverson D, Li IT, Stirnemann G,
Walker GC and Berne BJ. How osmolytes
influence hydrophobic polymer conformations: A
unified view from experiment and theory. Proc.
Natl. Acad. Sci. (2015) 30: 9270–5.
(35) Terry G, King F, Wiltsek C, Chu R and Cannon JG.
Evaluation two models for the effects of osmolytes
on protein stability and function: measuring the
interactions of Glycine betaine with carboxylic
acids. Ga. J. Sci. (2017) 75: 67.
(36) Jaworska K, Hering D, Mosieniak G, BielakZmijewska A, Pilz M, Konwerski M, Gasecka
A, Kapłon-Cieślicka A, Filipiak K, Sikora E and
Hołyst R. TMA, a forgotten uremic toxin, but not
TMAO, is involved in cardiovascular pathology.
Toxins (2019) 9: 490.
(37) Attri P, Venkatesu P, Kaushik N and Choi EH.
TMAO and sorbitol attenuate the deleterious action
of atmospheric pressure non-thermal jet plasma on
α-chymotrypsin. RSC Adv. (2012) 18: 7146–55.
(38) Ufnal M, Zadlo A and Ostaszewski R. TMAO: A
small molecule of great expectations. Nutrition
(2015) 13: 17–23.
(39) Wang J and Hui N. Zwitterionic poly (carboxybetaine)
functionalized conducting polymer polyaniline
nanowires for the electrochemical detection of
carcinoembryonic antigen in undiluted blood
serum. Bioelectrochemistry (2019) 125: 90–6.
(40) Adamczak B, Kogut M and Czub J. Effect of 
Mojtabavi S et al. / IJPR (2019), 18 (Special Issue): 13-30
osmolytes on the thermal stability of proteins:
replica exchange simulations of Trp-cage in urea
and betaine solutions. Phys. Chem. Chem. Phys.
(2018) 16: 11174–82.
(41) Su Y, Guo QQ, Wang S, Zhang X and Wang J.
Effects of betaine supplementation on l-threonine
fed-batch fermentation by Escherichia coli.
Bioprocess Biosyst. Eng. (2018) 10: 1509–18.
(42) Reslan M, Ranganathan V, Macfarlane DR and
Kayser V. Choline ionic liquid enhances the
stability of Herceptin® (trastuzumab). Chem.
Commun. (2018) 75:10622–5.
(43) Momeni L, Shareghi B, Farhadian S and Raisi F.
Making bovine trypsin more stable and active by
erythritol: A multispectroscopic analysis, docking
and computational simulation methods. J. Mol.
Liq. (2019) 292: 111389.
(44) Moosa MM, Ferreon JC and Ferreon AC. Ligand
interactions and the protein order-disorder
energetic continuum. Semin. Cell Dev. Biol. doi.
(45) Liu W, Wang M, Xu S, Gao C and Liu J. Inhibitory
effects of shell of Camellia oleifera Abel extract on
mushroom tyrosinase and human skin melanin. J.
Cosmet. Dermatol. (2019): 1–6.
(46) Jiang H, Liu GL, Chi Z, Hu Z and Chi ZM.
Genetics of trehalose biosynthesis in desertderived Aureobasidium melanogenum and role of
trehalose in the adaptation of the yeast to extreme
environments. Curr. Genet. (2018) 2: 479–91.
(47) Bertl A, Brantl V, Scherbaum N, Rujescu D and
Benninghoff J. Trehalose as glucose surrogate in
proliferation and cellular mobility of adult neural
progenitor cells derived from mouse hippocampus.
J. Neural Transm. (2019): 1–7.
(48) Meurs KM, Friedenberg SG, Kolb J, Saripalli C,
Tonino P, Woodruff K, Olby NJ, Keene BW, Adin
DB, Yost OL, DeFrancesco TC, Lahmers S, Tou S,
Shelton GD and Granzier H. A missense variant
in the titin gene in Doberman pinscher dogs with
familial dilated cardiomyopathy and sudden
cardiac death. Hum. Genet. (2019) 5: 515–24.
(49) Ho-Palma AC, Toro P, Rotondo F, Romero MD,
Alemany M, Remesar X and Fernández-López
JA. Insulin controls triacylglycerol synthesis
through control of glycerol metabolism and despite
increased lipogenesis. Nutrients (2019) 3: 513.
(50) Nayar R. inventor; Advanced Bioscience
Farmaceutica Ltda, assignee. Stable Factor VIII
formulations with low sugar-glycine. U.S. patent
(2018) 9,907,835.
(51) Anko M, Bjelošević M, Planinšek O, Trstenjak U,
Logar M, Grabnar PA and Brus B. The formation
and effect of mannitol hemihydrate on the stability
of monoclonal antibody in the lyophilized state.
Int. J. Pharm. Sci. (2019) 4: 106–16.
(52) Sun Y, Shen Z, Zhang C, Yi Y, Zhu K, Xu F and Kong
W. Development of a stable liquid formulation for
live attenuated influenza vaccine. J. Pharm. Sci.
(2019) 7: 2315–22.
(53) Hirao Y, Takechi K, Uriyu K and Uemura Y,
inventors; Green Cross Corp, assignee. Gammaglobulin injecTable solutions containing sorbitol.
U.S. patent (1989) 4,876,088.
(54) Bakaltcheva I, O’Sullivan AM, Hmel P and Ogbu
H. Freeze-dried whole plasma: evaluating sucrose,
trehalose, sorbitol, mannitol and glycine as
stabilizers. Thromb. Res. (2007) 1: 105–16.
(55) Ferrari SM, Elia G, Ragusa F, Paparo SR, Caruso
C, Benvenga S, Fallahi P and Antonelli A. The
protective effect of myo-inositol on human
thyrocytes. Rev. Endocr. Metab. Disord. (2018) 4:
(56) Bradbury SL and Jakoby WB. Glycerol as an
enzyme-stabilizing agent: effects on aldehyde
dehydrogenase. Proc. Natl. Acad. Sci. (1972) 9:
(57) Sun XB, Lim GT, Lee J, Wan JX, Lin HZ, Yang
JM, Wang Q and Park YD. Effects of osmolytes on
the refolding of recombinant Pelodiscus sinensis
brain-type creatine kinase. Process Biochem.
(2018) 68:83–92.
(58) Wlodarczyk SR, Custódio D, Pessoa Jr A and
Monteiro G. Influence and effect of osmolytes in
biopharmaceutical formulations. Eur. J. Pharm.
Biopharm. (2018) 31: 92–8.
(59) Kuhlmann AU, Hoffmann T, Bursy J, Jebbar M
and Bremer E. Ectoine and hydroxyectoine as
protectants against osmotic and cold stress: uptake
through the SigB-controlled betaine-cholinecarnitine transporter-type carrier EctT from
Virgibacillus pantothenticus. J. Bacteriol. (2011)
18: 4699–708.
(60) Lavrentyeva SI, Ivachenko LY, Golokhvast KS and
Nawaz MA. Ribonuclease activity of Glycine max
and Glycine soja sprouts as a marker adaptation
to copper sulphate and zinc sulphate toxicity.
Biochem. Syst. Ecol. (2019) 83: 66–70.
(61) Czech L, Hermann L, Stöveken N, Richter A,
Höppner A, Smits S, Heider J and Bremer E. Role
of the extremolytes ectoine and hydroxyectoine
as stress protectants and nutrients: genetics,
phylogenomics, biochemistry, and structural
analysis. Genes (2018) 4: 177.
(62) Smiatek J, Harishchandra RK, Rubner O, Galla
HJ and Heuer A. Properties of compatible solutes
in aqueous solution. Biophys. Chem. (2012) 160:
Osmolyte-Induced Folding and Stability of Proteins
(63) Abe Y, Ohkuri T, Yoshitomi S, Murakami S and
Ueda T. Role of the osmolyte taurine on the folding
of a model protein, hen egg white lysozyme, under
a crowding condition. Amino acids (2015) 5: 909–
(64) Rani A and Venkatesu P. Changing relations between
proteins and osmolytes: a choice of nature. Phys.
Chem. Chem. Phys. (2018) 31: 20315–33.
(65) Klenner D, Kleinhammer G and Deeg R, inventors;
Roche Diagnostics GmbH, assignee. Process for
stabilizing the activity of peroxidase in solution.
U.S. patent (1988) 4,757,016.
(66) Singh LR, Poddar NK, Dar TA, Rahman S, Kumar R
and Ahmad F. Forty years of research on osmolyteinduced protein folding and stability. J. Iran Chem.
Soc. (2011) 1: 1–23.
(67) Auton M, Bolen DW and Rösgen J. Structural
thermodynamics of protein preferential solvation:
osmolyte solvation of proteins, aminoacids, and
peptides. Proteins: Struct. Funct. Bioinf. (2008) 4:
(68) Fedotova MV. Compatible osmolytesbioprotectants: Is there a common link between
their hydration and their protective action under
abiotic stresses? J. Mol. Liq. (2019) 19: 111339.
(69) Harries D and Rösgen JA. Practical guide on how
osmolytes modulate macromolecular properties.
Methods Cell Biol. (2008) 1: 679–735.
(70) Rydeen AE, Brustad EM and Pielak GJ. Osmolytes
and Protein–Protein Interactions. J. Am. Chem.
Soc. (2018) 24: 7441–4.
(71) Monteith WB, Cohen RD, Smith AE, GuzmanCisneros E and Pielak GJ. Quinary structure
modulates protein stability in cells. Proc. Natl.
Acad. Sci. (2015) 6: 1739–42.
(72) Smith AE, Zhou LZ, Gorensek AH, Senske M and
Pielak GJ. In-cell thermodynamics and a new role
for protein surfaces. Proc. Natl. Acad. Sci. (2016)
7: 1725–30.
(73) Suraj Sharma G, Ali Dar T and Rajendrakumar
Singh L. Reshaping the protein folding pathway by
osmolyte via its effects on the folding intermediates.
Curr. Protein Pept. Sci. (2015) 16: 513–20.
(74) Rösgen J. Molecular basis of osmolyte effects
on protein and metabolites. In: Methods in
enzymology. Academic Press. (2007) 428: 459–86.
(75) Timasheff SN. Control of protein stability and
reactions by weakly interacting cosolvents: the
simplicity of the complicated. Adv. Protein Chem.
(1998) 51: 355–432.
(76) Wang Y, Sarkar M, Smith AE, Krois AS and Pielak
GJ. Macromolecular crowding and protein stability.
J. Am. Chem. Soc. (2012) 40: 16614–18.
(77) Bennion BJ, DeMarco ML and Daggett V. Preventing
misfolding of the prion protein by trimethylamine
N-oxide. Biochemistry (2004) 41: 12955–63.
(78) Kumar R. Role of naturally occurring osmolytes
in protein folding and stability. Arch. Biochem.
Biophys. )2009) 491: 1–6.
(79) Phillips JC, Braun R, Wang W, Gumbart J,
Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L
and Schulten K. Scalable molecular dynamics with
NAMD. J. Comput. Chem. (2005) 26: 1781–802.
(80) Dipaola G and Belleau B. Polyol–Water interactions.
Apparent molal heat capacities and volumes of
aqueous polyol solutions. Can. J. Chem. (1977)
22: 3825–30.
(81) Batchelor JD, Olteanu A, Tripathy A and Pielak
GJ. Impact of protein denaturants and stabilizers
on water structure. J. Am. Chem. Soc. (2004) 7:
(82) Back JF, Oakenfull D and Smith MB. Increased
thermal stability of proteins in the presence of
sugars and polyols. Biochemistry (1979) 18: 5191–
(83) Politi R, Sapir L and Harries D. The impact of polyols
on water structure in solution: a computational
study. J. Phys. Chem. A (2009) 26: 7548–55.
(84) Pincus DL, Hyeon C and Thirumalai D. Effects of
trimethylamine N-oxide (TMAO) and crowding
agents on the stability of RNA hairpins. J. Am.
Chem. Soc. (2008) 23: 7364–72.
(85) Bolen DW and Rose GD. Structure and energetics of
the hydrogen-bonded backbone in protein folding.
Annu. Rev. Biochem. (2008) 77: 339–62.
(86) Venkatesu P, Lee MJ and Lin HM. Trimethylamine
N-oxide counteracts the denaturing effects of
urea or GdnHCl on protein denatured state. Arch.
Biochem. Biophys. (2007) 1: 106–15.
(87) Lee JC and Timasheff SN. The stabilization of
proteins by sucrose. J. Biol. Chem. (1981) 14:
(88) Greenfield NJ. Determination of the folding of
proteins as a function of denaturants, osmolytes
or ligands using circular dichroism. Nat. Protoc.
(2006) 6: 2733.
(89) Celinski SA and Scholtz JM. Osmolyte effects on
helix formation in peptides and the stability of
coiled-coils. Protein Sci. (2002) 8: 2048–51.
(90) Greenfield NJ. Using circular dichroism spectra to
estimate protein secondary structure. Nat. Protoc.
(2006) 6: 2876.
(91) Mukaiyama A, Koga Y, Takano K and Kanaya S.
Osmolyte effect on the stability and folding of a
hyperthermophilic protein. Proteins: Struct. Funct.
Bioinf. (2008) 1: 110–8.
(92) Chowdhury PK. Fluorescence correlation
spectroscopy: a brief review of techniques and 
Mojtabavi S et al. / IJPR (2019), 18 (Special Issue): 13-30
applications to biomolecules and biosystems. J.
Proteins Proteomics (2013) 2.
(93) Jain S, Seechurn S, Gupta P, Garg G, Dhamija
B, Latha N and Yvr KS. Effects of osmolytes on
the structural stability of bovine trypsin: A brief
review. J. Pharm. Res. (2015) 8: 500–8.
(94) Sharma S, Pathak N and Chattopadhyay K.
Osmolyte induced stabilization of protein
molecules: a brief review. J. Proteins Proteomics
(2013) 10: 3(2).
(95) Plaza del Pino IM and Sanchez-Ruiz JM. An
osmolyte effect on the heat capacity change for
protein folding. Biochem. (1995) 27: 8621–30.
(96) Issaq HJ and Veenstra TD. Two-dimensional
polyacrylamide gel electrophoresis (2D-PAGE):
advances and perspectives. BioTechniques (2008)
5: 697–700.
(97) Bye JW, Platts L and Falconer RJ. Biopharmaceutical
liquid formulation: a review of the science
of protein stability and solubility in aqueous
environments. Biotechnol. Lett. (2014) 5: 869–75.
(98) Pradhan N, Jana NR and Jana NR. Inhibition of protein
aggregation by iron oxide nanoparticles conjugated
with glutamine-and proline-based osmolytes. ACS
Appl. Nano Mater. (2018) 3: 1094–10.