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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules
Physicochemical Insights into Stabilisation of Stressed Lysozyme and Glycine Homopeptides by Sorbitol Ritutama Ghosh, and Nand Kishore J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04394 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018
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The Journal of Physical Chemistry
Physicochemical Insights into Homopeptides by Sorbitol
Stabilisation of Stressed Lysozyme
and Glycine
Ritutama Ghosh, Nand Kishore* Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai – 400 076, India. *Corresponding author (Email:
[email protected]) _____________________________________________________________________________ ABSTRACT Understanding the mode of action of osmolytes on the protein with and without stressed conditions still requires experimental proof. In this direction we have studied the interactions of a model protein hen egg-white lysozyme (HEWL) and some homopeptides with sorbitol and mixture of [dodecyltrimethylammonium bromide (DTAB) + sorbitol] by using a combination of high sensitivity calorimetry, density, sound velocity and conductivity measurements with spectroscopic support. Physical chemistry underlying these interactions has been addressed based on energetics of interactions and other physicochemical properties. These results have highlighted that even though the number of groups increase in higher homopeptides, the hydrophobic effect of groups in the peptides dominates. Further, the counteraction of the deleterious effects of DTAB by sorbitol is by strengthened DTAB-sorbitol interactions rather than indirect effect of the osmolyte via preferential exclusion. The results provide insights into the nature of interactions of the protein as well as some of the building blocks with the (DTAB + osmolyte) mixture which helped in understanding mode of action of the osmolyte. Detailed physicochemical insights into the mode of action of stress counteracting agents on the protein and its destabiliser are needed to develop strategies to achieve optimum stability and activity of proteins under such conditions. _____________________________________________________________________________ 1. INTRODUCTION Osmolytes are small organic molecules which stabilise conformation of a protein. They help in protein folding by shifting the equilibrium of the reaction, Unfolded [U] Native [N].1 2-5 Osmolytes have been classified into molecules which act by direct mechanism and indirect mechanism.6-10 It was inferred by Bolen and co-workers that a mechanism known as “direct but unfavourable mechanism” exists.11-13 Osmolytes are also used in medical sciences and have been used in protecting renal cells by releasing hyperosmotic stress.14 Osmolytes such as betaine and myo-inositol have been used in the treatment of chronic kidney disease.14 Further, it is known that polyols like sorbitol, glycerol, mannitol, xylitol, adonitol and inositol are capable of performing hydrogen bonding and are preferentially excluded from the protein surface.15 Sorbitol has also been reported to play a major role in suppressing protein aggregation. It prevents self-aggregation of unfolded lysozyme stabilising the folded form of protein.16 The microenvironment of a protein is altered by various factors such as temperature, pressure, pH, additives and others. The stress response to such harsh conditions in protein affects 1 ACS Paragon Plus Environment
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its native conformation. Harsh environment also includes conditions such as heat, metal, surfactant, cellular stress, glutathionylated proteins in oxidative condition and partially unfolded state among others. Osmolyte as a co-solvent disturbs the protein-solvent interaction by affecting hydration properties thereby stabilising or destabilising the protein. The counteraction of stressed conditions can lead to regain of the conformational stability and activity of the protein. If the stress is by a denaturant, a co-solute which can reduce the effect of denaturant can help in achieving this condition. Surfactants are molecules having hydrophobic tails and hydrophilic head groups. The monomers arrange into clusters with a specific aggregation number at critical micellar concentration (CMC). At CMC, the spherical micelles are usually formed such that the hydrophobic tails reside inside and the polar head groups reside outside. The polar head groups being at the surface interact with the protein. Surfactants find applications in medical biology includes artificial implants, drug carriers, biomembranes, transfection, biolubrication, lipoproteins, pharmaceuticals, anaesthetics.17 In order to meet their usefulness avoiding harmful effects on proteins, stabilisers are introduced in the system. Osmolytes, being good stabilisers, can counteract the deleterious effects of surfactants. It is reported that sarcosine counteracts the effect of urea.10,18 The concentration of urea in mammalian kidney is found to be very high around (3-5) M.14 The hyperosmotic stress is met up by betaine and myo-inositol.14 Even though there are some reports available in literature which describe stabilisation of protein by sorbitol in the presence of urea,19,20 insights into the cause of stabilisation in terms of the energetics of the interaction are missing. It needs to be established whether the observed stabilisation is due to direct interaction with protein, solvent mediated effects or interaction with the stress causing agents. In this work we have addressed physical chemistry insights into the action of osmolyte sorbitol in providing conformational stability to the protein in the presence of stress causing agent dodecyltrimethylammonium bromide (DTAB). Physicochemical properties such as partial molar volume, partial molar compressibility, enthalpies of interaction provide useful information about the nature of solute-solvent interactions. In view of complexities in protein conformation, finer insights into interactions can be obtained from the model compound studies. The work reported in this manuscript analyses the interactions of the mixture of (DTAB + sorbitol) with peptides and protein and addresses the mode of action of osmolyte in counteracting the effect of denaturant based on energetics of interactions supported by conformational analysis. In this work we have chosen some homopeptides as building blocks of proteins and studied their interaction with mixture of (DTAB + sorbitol). The interactions of various amino acids and peptides in different solvents has provided valuable information about solute-solvent interactions.18,21-23 Further, the work has been extended to interaction of the mixture with the model protein hen egg white lysozyme. In order to understand whether the effect of osmolyte on the protein is direct or mediated via interaction with DTAB, efforts have been made to understand the interaction of the osmolyte with the monomers and micelles of the surfactant. Obtaining physical chemistry insights into interactions of stabilisers with the stress causing agents and the protein under stressed environment is essential to understand the mode of 2 ACS Paragon Plus Environment
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The Journal of Physical Chemistry
action and hence develop strategies to achieve optimum environment for the effective biological properties of the protein. The future of such studies lies in developing suitable additives or mixture of additives which can provide substantial stabilisation to the proteins under stress and hence maintaining their biological activities. 2. EXPERIMENTAL 2.1. Materials and methods. Glycine, diglycine, triglycine, pentaglycine, D-Sorbitol, henegg white lysozyme and potassium phosphate were purchased from Sigma–Aldrich Chemical Company, USA. Dodecyltrimethylammonium bromide (DTAB) was procured from Tokyo Chemical Industry Co. Ltd. (TCI), Japan. Glycine and its peptides were used without further purification after drying over P2O5 in a vacuum desiccator. Water, used to prepare all the solutions was double distilled. Prior to preparing the solutions for density and heats of dilution/interaction measurements, water was also thoroughly degassed. All of the mass determinations were executed on a Sartorius BP 211D digital balance which had a readability of ± 0.01 mg. Overnight dialysis of the stock protein solutions was done against phosphate buffer at 4oC with three changes of buffer. A Jasco V-550 double beam spectrophotometer was used for determination of concentration of the dialysed protein after centrifugation by using an extinction
% coefficient corresponding to = 26.50.24 2.2. Density and speed of sound measurements. The densities of the solutions were measured on DSA 5000 digital density and sound velocity analyzer purchased from Anton Paar GmbH, Austria. The chemical calibration of the densimeter was performed by measuring the values of density and adiabatic compressibility of aqueous sodium chloride solutions at different molalities, which showed excellent agreement with a precision of ± 0.02 cm3 mol-1 in volume and approximately 3 MHz in working frequency for the speed of sound.25 The values of apparent molar volume (,ø) and apparent molar adiabatic compressibility (,,∅) of glycine and glycyl peptides in 0.100 mol kg-1 equimolal (DTAB + sorbitol) aqueous solution at T = 298.15 K were determined, respectively from the density () and isentropic compressibility ( ) of the solution by using the following equations,
,ø = ,,∅ =
( )
#$
(1)
"
(#$ #$ )
(2)
"
Here, is density of the reference solvent in g cm-3, % is molality of the solution in mol kg , & is molar mass of the solute in g mol-1, and ' is adiabatic compressibility of the reference solvent in Pa-1. The value of adiabatic compressibility of the aqueous solution was calculated from the speed of sound ( by using, -1
= * . )
(3)
2.3. Isothermal titration calorimetry. Isothermal titration calorimetric (ITC) measurements were performed at T = 298.15 K on a Nano ITC (TA Instruments, New Castle, DE, USA). The 250 µL syringe of the ITC was filled with aqueous glycine, glycyl homopeptide or protein 3 ACS Paragon Plus Environment
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solutions and titrated at a stirring speed of 250 rpm in all the experiments. The sample cell of 940 µL capacity was filled with 0.100 mol kg-1 aqueous solution of (DTAB + sorbitol) mixture. The reference cell was filled with water. The molalities of glycine, diglycine, triglycine and pentaglycine taken in the syringe were 0.200, 0.025, 0.025 and 0.003 mol kg-1, respectively. For studies on the interaction of the protein with surfactant and osmolyte, lysozyme was taken in the syringe and titrated into aqueous solution of DTAB and sorbitol taken in the cell individually, or in the mixture. A total of 25 consecutive injections of sample from the syringe were made of 20 s duration each and with a 240 s interval between successive injections. The ITC experiments provided a set of values of heat evolved or absorbed as a result of interactions. The values of ' standard molar enthalpy of interaction (∆" ) of glycine, glycyl homopeptide solutions with protein was determined by fitting the following equation to the values of the measured heats (,), ' , = ∆" + ./ %
(4)
Here % is molality of the solution and ./ is the empirical slope in J mol-2 kg. 2.4. Conductivity measurements. Autoranging Conductivity / TDS meter TCM 15+, with a temperature compensation cell having cell constant of 1.086 cm-1 was used to perform the conductivity measurements. The conductometer was calibrated for its cell constant by using aqueous KCl solutions. The conductance values were accurate within 0.5 %. 1.000 mol kg-1 DTAB solution was titrated into water; 0.100 mol kg-1 sorbitol; (0.100 mol kg-1 sorbitol + 0.200 mol kg-1 glycine); (0.100 mol kg-1 sorbitol + 0.025 mol kg-1 diglycine); (0.100 mol kg-1 sorbitol + 0.025 mol kg-1 triglycine); (0.100 mol kg-1 sorbitol + 0.003 mol kg-1 pentaglycine) and (0.100 mol kg-1 sorbitol + 0.08×10-3 mol dm-3 lysozyme) individually. A total of 27 consecutive injections with 10 µl each of 1.000 mol kg-1 DTAB were made. The experiments were carried out at 298 K, 303 K, 308 K and 313 K maintaining the temperatures (± 0.1 K) by using ColeParmer circulating thermostat. The values of critical micelle concentration (CMC or c*) of the surfactants were obtained from the change in slope of the measured conductance values plotted ' against DTAB concentration. The values of enthalpy of micellisation ( ∆"01 ), entropy of ' ' micellisation (∆."01 ) and Gibb’s free energy change of micellisation (∆2"01 ) were calculated by using the following equations,26,27 ' ∆"01 " = (1 + 4)56 ×
89: (1∗)
(5)
8
