Analysis of cold denaturation mechanism of β-lactoglobulin and comparison with thermal denaturation from Raman spectroscopy investigations

Cold- and heat-induced β-lactoglobulin (BLG) transformations have been analyzed in the presence of 4 M urea, from Raman spectroscopy investigations carried out simultaneously in the low wavenumber range (10–400 cm−1) and in the amide I region (1500–1800 cm−1). These investigations show common features between the denaturation processes at low and high temperatures. The denatured states are reached via an intermediate state characterized by a soft tertiary structure without detectable conformational changes. This intermediate is intimately connected with a tetrahedral hydrogen-bond structure of water which extends over a limited range. It is shown that the disruption of the hydrogen-bond network of D2O has an important consequence on the solvent dynamics, which controls protein dynamics and is characterized by an anharmonic behavior. By monitoring the amide I mode, conformational changes are detected at low temperature (below 5 °C) and determined to be similar to those detected at high temperature in the presence of urea near 65 °C, and in the absence of urea near 80 °C. The conformational changes are described as a loss of α-helix structures and a concomitant formation of β-sheets. The temperature dependence of the amide I wavenumber in BLG dissolved in the 4 M urea aqueous solution was interpreted on the basis of a two-state model, leading to the protein stability curve related to its molecular conformation. Copyright © 2011 John Wiley & Sons, Ltd.

[1]  G. Walrafen.  Raman Spectral Studies of the Effects of Urea and Sucrose on Water Structure , 1966 .

[2]  N. Pace,et al.  Thermodynamics of the unfolding of beta-lactoglobulin A in aqueous urea solutions between 5 and 55 degrees. , 1968, Biochemistry.

[3]  L. Genzel,et al.  Low‐frequency Raman spectra of lysozyme , 1976, Biopolymers.

[4]  A. Dunker,et al.  Determination of the secondary structure of proteins from the amide I band of the laser Raman spectrum. , 1981, Journal of molecular biology.

[5]  G. Klarenbeek,et al.  A differential scanning calorimetric study of the thermal behaviour of bovine β-lactoglobulin at temperatures up to 160 °C , 1981, Journal of Dairy Research.

[6]  P. Hegg,et al.  Thermal stability of whey proteins studied by differential scanning calorimetry , 1985 .

[7]  M. Bellissent-Funel,et al.  Spatial Correlations in Deeply Supercooled Water , 1986 .

[8]  G. Walrafen,et al.  Temperature dependence of the low‐ and high‐frequency Raman scattering from liquid water , 1986 .

[9]  H. Friedman,et al.  The thermodynamics of protein stability. Cold destabilization as a general phenomenon. , 1988, Biophysical chemistry.

[10]  C M Dobson,et al.  Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin. , 1989, Biochemistry.

[11]  K. Kuwajima,et al.  The molten globule state as a clue for understanding the folding and cooperativity of globular‐protein structure , 1989, Proteins.

[12]  F. Franks,et al.  The cold‐induced denaturation of lactate dehydrogenase at sub‐zero temperatures in the absence of perturbants , 1989, FEBS letters.

[13]  P. Privalov,et al.  Cold Denaturation of Protein , 1990 .

[14]  S Cusack,et al.  Temperature dependence of the low frequency dynamics of myoglobin. Measurement of the vibrational frequency distribution by inelastic neutron scattering. , 1990, Biophysical journal.

[15]  F. Franks,et al.  Stability of proteins at subzero temperatures: thermodynamics and some ecological consequences , 1991 .

[16]  P. Privalov,et al.  Calorimetric study of the heat and cold denaturation of beta-lactoglobulin. , 1992, Biochemistry.

[17]  S. Nakai,et al.  Raman spectroscopic study of thermally induced gelation of whey proteins , 1993 .

[18]  H. Mantsch,et al.  Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. , 1993, Biochemistry.

[19]  V. Kutyshenko,et al.  Differences in the processes of beta-lactoglobulin cold and heat denaturations. , 1994, Biophysical journal.

[20]  O. Nielsen,et al.  Low-frequency Raman spectroscopy , 1995 .

[21]  A. Jonas,et al.  NMR study of the cold, heat, and pressure unfolding of ribonuclease A. , 1995, Biochemistry.

[22]  G. Walrafen,et al.  Low-Frequency Raman Scattering from Water at High Pressures and High Temperatures† , 1996 .

[23]  P. Privalov,et al.  Intermediate states in protein folding. , 1996, Journal of molecular biology.

[24]  X L Qi,et al.  Effect of temperature on the secondary structure of beta-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis. , 1997, The Biochemical journal.

[25]  K. Hinsen,et al.  A simplified force field for describing vibrational protein dynamics over the whole frequency range , 1999 .

[26]  M. Karplus,et al.  Analysis of Calculated Normal Modes of a Set of Native and Partially Unfolded Proteins , 1999 .

[27]  R. Winter,et al.  Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering. , 1999, Biochemistry.

[28]  T. Norisuye,et al.  Heat-Induced Gelation of β-Lactoglobulin. 1. Time-Resolved Dynamic Light Scattering , 2000 .

[29]  K. Kawano,et al.  Stability of the molten globule state of a domain-exchanged chimeric protein between human and bovine alpha-lactalbumins. , 2000, Protein engineering.

[30]  F. Meersman,et al.  Pressure-assisted cold unfolding of proteins and its effects on the conformational stability compared to pressure and heat unfolding , 2000 .

[31]  S. Magazù,et al.  Destructuring effect of trehalose on the tetrahedral network of water: a Raman and neutron diffraction comparison , 2002 .

[32]  K. Wynne,et al.  Low-frequency modes of peptides and globular proteins in solution observed by ultrafast OHD-RIKES spectroscopy. , 2003, Biophysical journal.

[33]  J. Martí,et al.  An interpretation of the low-frequency spectrum of liquid water , 2003 .

[34]  Walraj S. Gosal,et al.  Fibrillar β-Lactoglobulin Gels: Part 1. Fibril Formation and Structure , 2004 .

[35]  Xiaolin Tang,et al.  Freeze-Drying Process Design by Manometric Temperature Measurement: Design of a Smart Freeze-Dryer , 2005, Pharmaceutical Research.

[36]  P. Bordat,et al.  Influence of homologous disaccharides on the hydrogen-bond network of water: complementary Raman scattering experiments and molecular dynamics simulations. , 2005, Carbohydrate research.

[37]  M. Pikal,et al.  The Effect of Stabilizers and Denaturants on the Cold Denaturation Temperatures of Proteins and Implications for Freeze-Drying , 2005, Pharmaceutical Research.

[38]  P. Bordat,et al.  Sugar bioprotective effects on thermal denaturation of lysozyme: Insights from Raman scattering experiments and molecular dynamics simulation , 2006 .

[39]  M. Descamps,et al.  Evidence of a two-stage thermal denaturation process in lysozyme: a Raman scattering and differential scanning calorimetry investigation. , 2006, The Journal of chemical physics.

[40]  M. Descamps,et al.  Microscopic description of protein thermostabilization mechanisms with disaccharides from Raman spectroscopy investigations , 2007 .

[41]  A. Morresi,et al.  Hydrogen bond dynamics and water structure in glucose-water solutions by depolarized Rayleigh scattering and low-frequency Raman spectroscopy. , 2007, The Journal of chemical physics.

[42]  M. Descamps,et al.  Thermostabilization mechanism of bovine serum albumin by trehalose. , 2009, The journal of physical chemistry. B.

[43]  P. Bordat,et al.  Low-frequency vibrational properties of lysozyme in sugar aqueous solutions: a Raman scattering and molecular dynamics simulation study. , 2009, The Journal of chemical physics.

[44]  M. Descamps,et al.  Thermal denaturation of beta-lactoglobulin and stabilization mechanism by trehalose analyzed from Raman spectroscopy investigations. , 2010, The journal of physical chemistry. B.

[45]  Slowing down of water dynamics in disaccharide aqueous solutions , 2010, 1005.5327.