Domain-Specific Stabilization of Structural and Dynamic Responses of Human Serum Albumin by Sucrose

Author(s): Vaisakh Mohan, Bhaswati Sengupta, Nilimesh Das, Indrani Banerjee, Pratik Sen*.

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 4 , 2019

Submit Manuscript
Submit Proposal

Graphical Abstract:


Abstract:

Background: Human Serum Albumin (HSA) is the most abundant protein present in human blood plasma. It is a large multi-domain protein with 585 amino acid residues. Due to its importance in human body, studies on the interaction of HSA with different external agent is of vital interest. The denaturation and renaturation of HSA in presence of external agents are of particular interest as they affect the biological activity of the protein.

Objective: The objective of this work is to study the domain-specific and overall structural and dynamical changes occurring to HSA in the presence of a denaturing agent, urea and a renaturing agent, sucrose.

Methods: In order to carry out the domain-specific studies, HSA has been tagged using N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) at Cys-34 of domain-I and pnitrophenyl coumarin ester (NPCE) at Tyr-411 site in domain-III, separately. Steady-state absorption, emission and solvation dynamic measurements have been carried out in order to monitor the domain-specific alteration of HSA caused by the external agents. The overall structural change of HSA have been monitored using circular dichroism spectroscopy.

Results: The α-helicity of HSA was found to decrease from 65% to 11% in presence of urea and was found to further increase to 25% when sucrose is added, manifesting the denaturing and renaturing effects of urea and sucrose, respectively. The steady state studies show that domain-III is more labile towards denaturation as compared to domain-I. The presence of an intermediate state is observed during the denaturation process. The stabilization of this intermediate state in presence of sucrose is attributed as the reason for the stabilization of HSA by sucrose. From solvation dynamics studies, it could be seen that the solvation time of DACIA inside domain-I of HSA decreases and increases regularly with increasing concentrations of urea and sucrose, respectively, while in the case of NPCE-tagged domain-III, the effect of sucrose on solvation time is evident only at high concentrations of urea.

Conclusion: The denaturing and renaturing effects of urea and sucrose could be clearly seen from the steady state studies and circular dichroism spectroscopy measurements. A regular change in solvation time could only be observed in the case of domain-I and not in domain-III. The results indicate that the renaturing effect of sucrose on domain-III is not very evident when protein is in its native state, but is evident in when the protein is denatured.

Keywords: Human serum albumin, domain, denaturation, sucrose, protein stabilization, solvation dynamics.

[1]
Berg, J.; Tymoczko, J.; Stryer, L. Biochemistry, 5th ed; W.H. Freeman and company: New York, USA, 2002.
[2]
Nelson, D.; Cox, M. Lehninger Principles of Biochemistry, 5th ed; W.H. Freeman and company: New York, USA, 2008.
[3]
Pace, C.N.; Treviño, S.; Prabhakaran, E.; Scholtz, J.M. Protein structure, stability and solubility in water and other solvents. Philos. Trans. R. Soc. B Biol. Sci., 2004, 359(1448), 1225-1235.
[4]
Gromiha, M.M. Protein Bioinformatics from Sequence to Function, 1st ed; Elsevier Inc.: New Delhi, India, 2010.
[5]
Vorobjev, Y.N.; Hermans, J. Free energies of protein decoys provide insight into determinants of protein stability. Protein Sci., 2001, 10, 2498-2506.
[6]
Reece, J.B.; Campbell, N.A.; Myers, N.; Urry, L.A.; Cain, M.L.; Wasserman, S.A.; Minorsky, P.V.; Jackson, R.B.; Cooke, B.N. Campbell Biology; Pearson Education Australia: Sydney, 2011.
[7]
López-Alonso, J.P.; Bruix, M.; Font, J.; Ribó, M.; Vilanova, M.; Jiménez, M.A.; Santoro, J.; González, C.; Laurents, D.V. NMR Spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: Implications for oligomer formation by 3D domain swapping. J. Am. Chem. Soc., 2010, 132, 1621-1630.
[8]
Sawyer, W.H.; Puckridge, J. The dissociation of proteins by chaotropic salts. J. Biol. Chem., 1973, 248, 8429-8433.
[9]
Bhuyan, A.K. On the mechanism of SDS-induced protein denaturation. Biopolymers, 2009, 93, 186-199.
[10]
Scholtz, J.M.; Baldwin, R.L. Perchlorate-induced denaturation of ribonuclease A: Investigation of possible folding intermediates. Biochemistry, 1993, 32, 4604-4608.
[11]
Leggio, C.; Galantini, L.; Konarev, P.V.; Pavel, N.V. Urea-induced denaturation process on defatted human serum albumin and in the presence of palmitic acid. J. Phys. Chem. B, 2009, 113, 12590-12602.
[12]
Vagenende, V.; Yap, M.G.S.; Trout, B.L. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry, 2009, 48, 11084-11096.
[13]
Abe, M.; Abe, Y.; Ohkuri, T.; Mishima, T.; Monji, A.; Kanba, S.; Ueda, T. Mechanism for retardation of amyloid fibril formation by sugars in Vλ6 protein. Protein Sci., 2013, 22, 467-474.
[14]
Kumar, N.; Kishore, N. Protein stabilization and counteraction of denaturing effect of urea by glycine betaine. Biophys. Chem., 2014, 189, 16-24.
[15]
Wang, Y.; Sarkar, M.; Smith, A.E.; Krois, A.S.; Pielak, G.J. Macromolecular crowding and protein stability. J. Am. Chem. Soc., 2012, 134, 16614-16618.
[16]
Cottone, G. A Comparative study of carboxy myoglobin in saccharide-water systems by molecular dynamics simulation. J. Phys. Chem. B, 2007, 111, 3563-3569.
[17]
He, X.M.; Carter, D.C. Atomic structure and chemistry of human serum albumin. Nature, 1992, 358, 209.
[18]
Dockal, M.; Carter, D.C.; Rüker, F. The three recombinant domains of human serum albumin: Structural characterization and ligand binding properties. J. Biol. Chem., 1999, 274, 29303-29310.
[19]
Ghuman, J.; Zunszain, P.A.; Petitpas, I.; Bhattacharya, A.A.; Otagiri, M.; Curry, S. Structural basis of the drug-binding specificity of human serum albumin. J. Mol. Biol., 2005, 353, 38-52.
[20]
Yamasaki, K.; Chuang, V.T.G.; Maruyama, T.; Otagiri, M. Albumin-drug interaction and its clinical implication. Biochim. Biophys. Acta, Gen. Subj., 2013, 1830, 5435-5443.
[21]
Li, S.; Zhao, X.; Mo, Y.; Cummings, P.T.; Heller, W.T. Human serum albumin interactions with C60 fullerene studied by spectroscopy, small-angle neutron scattering, and molecular dynamics simulations. J. Nanopart. Res., 2013, 15, 1769.
[22]
Abou-Zied, O.K. Investigating 2,2‘-Bipyridine-3,3‘-Diol as a microenvironment-sensitive probe: Its binding to cyclodextrins and human serum albumin. J. Phys. Chem. B, 2007, 111, 9879-9885.
[23]
Jana, S.; Dalapati, S.; Ghosh, S.; Guchhait, N. Study of microheterogeneous environment of protein human serum albumin by an extrinsic fluorescent reporter: A spectroscopic study in combination with molecular docking and molecular dynamics simulation. J. Photochem. Photobiol. Bol. Biol., 2012, 112, 48-58.
[24]
Singh, R.B.; Mahanta, S.; Bagchi, A.; Guchhait, N. Interaction of human serum albumin with charge transfer probe ethyl ester of N,N-Dimethylamino Naphthyl Acrylic acid: An extrinsic fluorescence probe for studying protein micro-environment. Photochem. Photobiol. Sci., 2009, 8, 101-110.
[25]
Das, N.; Sen, P. Structural, functional, and dynamical responses of a protein in a restricted environment imposed by macromolecular crowding. Biochemistry, 2018, 57, 6078-6089.
[26]
González-Jiménez, J.; Cortijo, M. Urea-induced denaturation of human serum albumin labeled with acrylodan. J. Protein Chem., 2002, 21, 75-79.
[27]
Flora, K.; Brennan, J.D.; Baker, G.A.; Doody, M.A.; Bright, F.V. Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods. Biophys. J., 1998, 75, 1084-1096.
[28]
Shaw, A.K.; Pal, S.K. Spectroscopic studies on the effect of temperature on ph-induced folded states of human serum albumin. J. Photochem. Photobiol. Bol. Biol., 2008, 90, 69-77.
[29]
Wallewik, K. Reversible temperature, denaturation of human serum albumin and guanidine hydrochloride followed by optical rotation. J. Biol. Chem., 1973, 248, 2650-2655.
[30]
Wetzel, R.; Becker, M.; Behlke, J.; Billwitz, H.; Böhm, S.; Ebert, B.; Hamann, H.; Krumbiegel, J.; Lassmann, G. Temperature behaviour of human serum albumin. Eur. J. Biochem., 1980, 104, 469-478.
[31]
Abou-zied, O.K.; Al-Shihi, O.I.K. Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes. J. Am. Chem. Soc., 2008, 130, 10793-10801.
[32]
Picó, G.A. Thermodynamic features of the thermal unfolding of human serum albumin. Int. J. Biol. Macromol., 1997, 20, 63-73.
[33]
Krishnakumar, S.S.; Panda, D. Spatial relationship between the prodan site, Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding. Biochemistry, 2002, 41, 7443-7452.
[34]
Ahmad, B.; Khan, M.K.A.; Haq, S.K.; Khan, R.H. Intermediate formation at lower urea concentration in “B” isomer of human serum albumin: A case study using domain specific ligands. Biochem. Biophys. Res. Commun., 2004, 314, 166-173.
[35]
Ahmad, B.; Ahmed, M.Z.; Haq, S.K.; Khan, R.H. Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III. Biochim. Biophys. Acta. Proteins Proteomics, 2005, 1750, 93-102.
[36]
Ahmad, B. Ankita; Khan, R.H. Urea Induced unfolding of f isomer of human serum albumin: A case study using multiple probes. Arch. Biochem. Biophys., 2005, 437, 159-167.
[37]
Galantini, L.; Leggio, C.; Pavel, N.V. Human serum albumin unfolding: A small-angle x-ray scattering and light scattering study. J. Phys. Chem. B, 2008, 112, 15460-15469.
[38]
Del Giudice, A.; Leggio, C.; Balasco, N.; Galantini, L.; Pavel, N.V. Ibuprofen and propofol cobinding effect on human serum albumin unfolding in urea. J. Phys. Chem. B, 2014, 118, 10043-10051.
[39]
Del Giudice, A.; Dicko, C.; Galantini, L.; Pavel, N.V. Time-dependent PH scanning of the acid-induced unfolding of human serum albumin reveals stabilization of the native form by palmitic acid binding. J. Phys. Chem. B, 2017, 121, 4388-4399.
[40]
Heller, W.T. Comparison of the thermal denaturing of human serum albumin in the presence of guanidine hydrochloride and 1-butyl-3-methylimidazolium ionic liquids. J. Phys. Chem. B, 2013, 117, 2378-2383.
[41]
Zhuo, W.; Peng, X.; Lin, X. Insights into the interaction mechanism between tiagabine hydrochloride and two serum albumins. RSC Adv., 2018, 8, 24953-24960.
[42]
Mohan, V.; Sengupta, B.; Acharyya, A.; Yadav, R.; Das, N.; Sen, P. Region-specific double denaturation of human serum albumin : Combined effects of temperature and GnHCl on structural and dynamical responses. ACS Omega, 2018, 3, 10406-10417.
[43]
Muzammil, S.; Kumar, Y.; Tayyab, S. Anion‐induced stabilization of human serum albumin prevents the formation of intermediate during urea denaturation. Proteins Struct. Funct. Bioinform., 2000, 40, 29-38.
[44]
Yadav, R.; Sen, P. Mechanistic investigation of domain specific unfolding of human serum albumin and the effect of sucrose. Protein Sci., 2013, 22, 1571-1581.
[45]
Wang, R.; Sun, S.; Bekos, E.J.; Bright, F.V. Dynamics surrounding Cys-34 in native, chemically denatured, and silica-adsorbed bovine serum albumin. Anal. Chem., 1995, 67, 149-159.
[46]
Sengupta, B.; Acharyya, A.; Sen, P. Elucidation of the local dynamics of domain-III of human serum albumin over the Ps-[Small Mu ]s time regime using a new fluorescent label. Phys. Chem. Chem. Phys., 2016, 18, 28548-28555.
[47]
Zhong, D.; Pal, S.K.; Zewail, A.H. Biological water: A critique. Chem. Phys. Lett., 2011, 503, 1-11.
[48]
Jungwirth, P. Biological water or rather water in biology? J. Phys. Chem. Lett., 2015, 6, 2449-2451.
[49]
Bellissent-Funel, M-C.; Hassanali, A.; Havenith, M.; Henchman, R.; Pohl, P.; Sterpone, F.; van der Spoel, D.; Xu, Y.; Garcia, A.E. Water determines the structure and dynamics of proteins. Chem. Rev., 2016, 116, 7673-7697.
[50]
Nandi, N.; Bagchi, B. Dielectric relaxation of biological water. J. Phys. Chem. B, 1997, 101, 10954-10961.
[51]
Kamal, J.K.A.; Zhao, L.; Zewail, A.H. Ultrafast hydration dynamics in protein unfolding: Human serum albumin. Proc. Natl. Acad. Sci. USA, 2004, 101, 13411-13416.
[52]
Das, D.K.; Mondal, T.; Mandal, U.; Bhattacharyya, K. Probing deuterium isotope effect on structure and solvation dynamics of human serum albumin. ChemPhysChem, 2011, 12, 814-822.
[53]
Chowdhury, R.; Sen Mojumdar, S.; Sen, Chattoraj S.; Bhattacharyya, K. Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: Solvation dynamics study. J. Chem. Phys., 2012, 137, 55104.
[54]
Bagchi, B. Water dynamics in the hydration layer around proteins and micelles. Chem. Rev., 2005, 105, 3197-3219.
[55]
Mondal, S.; Mukherjee, S.; Bagchi, B. Origin of diverse time scales in the protein hydration layer solvation dynamics: A simulation study. J. Chem. Phys., 2017, 147, 154901-1-11.
[56]
Ben Ishai, P.; Tripathi, S.R.; Kawase, K.; Puzenko, A.; Feldman, Y. What is the primary mover of water dynamics? Phys. Chem. Chem. Phys., 2015, 17, 15428-15434.
[57]
Popov, I.; Ben Ishai, P.; Ben Khamzin, A.; Feldman, Y. The mechanism of the dielectric relaxation in water. Phys. Chem. Chem. Phys., 2016, 18, 13941-13953.
[58]
Kurzweil-Segev, Y.; Popov, I.; Eisenberg, I.; Yochelis, S.; Keren, N.; Paltiel, Y.; Feldman, Y. Confined water dynamics in a hydrated photosynthetic pigment-protein complex. Phys. Chem. Chem. Phys., 2017, 19, 28063-28070.
[59]
Yadav, R.; Sengupta, B.; Sen, P. Effect of sucrose on chemically and thermally induced unfolding of domain-I of human serum albumin: Solvation dynamics and fluorescence anisotropy Study. Biophys. Chem., 2016, 211, 59-69.
[60]
Maroncelli, M.; Fleming, G.R. Picosecond solvation dynamics of coumarin 153: The importance of molecular aspects of solvation. J. Chem. Phys., 1987, 86, 6221-6239.
[61]
Shil, S.; Sengupta, B.; Das, N.; Sen, P. Sucrose-induced stabilization of domain-II and Overall human serum albumin against chemical and thermal denaturation. ACS Omega, 2018, 3, 16633-16642.
[62]
Fee, R.S.; Maroncelli, M. Estimating the time-zero spectrum in time- resolved emission measurements of solvation dynamics. Chem. Phys., 1994, 183, 235.
[63]
Naidu, K.T.; Prabhu, N.P. Protein-surfactant interaction: Sodium dodecyl sulfate-induced unfolding of ribonuclease A. J. Phys. Chem. B, 2011, 115, 14760-14767.


Rights & PermissionsPrintExport Cite as


Article Details

VOLUME: 26
ISSUE: 4
Year: 2019
Page: [287 - 300]
Pages: 14
DOI: 10.2174/0929866526666190122115702
Price: $58

Article Metrics

PDF: 26
HTML: 5