Karel Heremans - Academia.edu (original) (raw)

Papers by Karel Heremans

Research paper thumbnail of General discussion

Faraday Discussions of The Chemical Society, 1982

Research paper thumbnail of Effect of high pressure on the heat activation in vivo of trehalase in the spores of Phycomyces blakesleeanus

European journal of biochemistry / FEBS, 1980

The effect of pressure on the heat activation in vivo of trehalase in the spores of Phycomyces bl... more The effect of pressure on the heat activation in vivo of trehalase in the spores of Phycomyces blakesleeanus has been investigated in order to obtain information about the molecular mechanism of the activation. For a protein conformational change directly induced in the enzyme by the heat treatment an upward shift with about 2-6 K/1000 atm (1.013 X 10(5) kPa) is to be expected in the moderate high-pressure region. On the other hand, for a phospholipid phase transition causing the activation, a continuous upward shift with about 20 K/1000 atm is to be expected. For trehalase activation we find a continuous upward shift of the activation temperature with about 5-9 K/1000 atm. The denaturation of trehalase, which occurs at slightly higher temperatures, is influenced by pressure completely as expected for a protein conformational change. The application of high pressure during spore heat activation makes it possibe to break the dormancy of the spores without concomitant activation of tr...

Research paper thumbnail of Piezotropic unfolding of lysozyme in pure D2O at the outer edge of excess hydration

This Letter reports a FTIR study on the pressure-induced unfolding of lysozyme in pure D 2 O clos... more This Letter reports a FTIR study on the pressure-induced unfolding of lysozyme in pure D 2 O close to the conditions of non excess hydration. An essential population of the proteins apparently refold into its native structure after pressure release. It could furthermore be shown that down to a hydration of h = 2 (h = m prot /m D 2 O ; m = mass), the unfolding pressure did not vary with hydration. Hydration dependent behaviour was found with respect to the change of the wavenumbers of the a-helical structure during unfolding. This result is discussed with respect to the reversibility of unfolding in pure solvents and with the effects of hydrogen exchange.

Research paper thumbnail of Pressure-Temperature Effects on Proteins: Unfolding, Aggregation and Fibrillogenesis

Defect and Diffusion Forum, 2002

Research paper thumbnail of Two-dimensional Fourier-transform infrared correlation spectroscopy study of the high-pressure tuning of proteins

Vibrational Spectroscopy, 2000

Generalized two-dimensional (2D) correlation spectroscopy and evolving factor analysis (EFA) have... more Generalized two-dimensional (2D) correlation spectroscopy and evolving factor analysis (EFA) have been applied to the study of cationic curing reaction of mixtures of diglycidyl ether of bisphenol A (DGEBA) and c-valerolactone (c-VL). The reaction has been monitored by Fourier transform infrared spectroscopy. The reaction periods in which a chemical change takes place are identified using EFA and then the sequence of changes are established by means of the interpretation of the synchronous and asynchronous spectra obtained with 2D correlation spectroscopy. By combining this information, in the curing process, four reactions have been detected:

Research paper thumbnail of 2D FT-IR spectroscopy analysis of the pressure-induced changes in proteins

Vibrational Spectroscopy, 1999

In this paper, we apply for the first time two-dimensional (2D) correlation spectroscopy to analy... more In this paper, we apply for the first time two-dimensional (2D) correlation spectroscopy to analyze pressure-induced changes in proteins. We show that it is possible to distinguish between hydrogen-deuterium (H/D) exchange and conformational changes from the synchronous and the asynchronous spectra. From the sequence of the spectral changes it can be concluded that the initial partial unfolding of the secondary

Research paper thumbnail of Compressibility of the Heme Pocket of Substrate Analogue Complexes of Cytochrome P -450cam-CO. The Effect of Hydrostatic Pressure on the Soret Band

European Journal of Biochemistry, 1995

The effect of hydrostatic pressure on the electronic absorption spectrum of the carbon monoxide c... more The effect of hydrostatic pressure on the electronic absorption spectrum of the carbon monoxide complex of cytochrome P-450cam (CYP101) in the presence of various substrates was studied.

Research paper thumbnail of Pressure-Tuning the Conformation of Bovine Pancreatic Trypsin Inhibitor Studied by Fourier-Transform Infrared Spectroscopy

European Journal of Biochemistry, 1996

A hydrostatic pressure of 1 .5 GPa induces changes in the secondary structure of bovine pancreati... more A hydrostatic pressure of 1 .5 GPa induces changes in the secondary structure of bovine pancreatic tryspin inhibitor (BPTI) as revealed by the analysis of the amide I' band with Fourier-transform infrared (FTIR) spectroscopy in the diamond anvil cell. The features of the secondary structure remain distinct at high pressure suggesting that the protein does not unfold. The fitted percentages of the secondary structure elements during compression and decompression strongly suggest that the pressure-induced changes are reversible. The pressure-induced changes in the tyrosine side chain band are also reversible. The results demonstrate that the infrared technique explores different aspects of the behaviour of proteins in comparison with two published molecular dynamics studies performed up to 1

Research paper thumbnail of Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin

European Journal of Biochemistry, 1999

Research paper thumbnail of Effect of High Pressure, Detergents and Phaospholipase on the Break in the Arrhenius Plot of Azotobacter Nitrogenase

European Journal of Biochemistry, 1978

It is shown that lipids are responsible for the breaks in the Arrhenius plots of Azotohuctr~r nit... more It is shown that lipids are responsible for the breaks in the Arrhenius plots of Azotohuctr~r nitrogenase. The physical evidence is that temperature at which the break occurs incrcases with increasing pressure by 20 KjlOOO atm. This is in agreement with the pressurc dependence of the transition temperature of several synthetic phospholipids. We also find the same pressure dependence for the broad transitions observed in Eschericlzia coli phosphatidylethanolamine and in the membrane lipids from Azotobucter itself. Detergents and phospholipase remove the break. Reconstruction can be performed only with specific phospholipids.

Research paper thumbnail of Intermolecular Interactions of Proteins under Pressure. Aggregation, Dissociation, Chaperoning

Defect and Diffusion Forum, 2002

Research paper thumbnail of Molecular mechanisms of pressure induced conformational changes in BPTI

Proteins: Structure, Function, and Genetics, 1996

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Research paper thumbnail of Extreme heat‐ and pressure‐resistant 7‐kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single‐point amino acid substitution

Proteins: Structure, Function, and Genetics, 1997

This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfa... more This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability. As observed in the NMR, FTIR, and CD spectra, wild-type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar. The pressure-induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented. These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance.

Research paper thumbnail of High pressure effects on protein structure and function

Proteins: Structure, Function, and Genetics, 1996

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and... more Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity. 0 1996 Wiley-Liss, Inc.

Research paper thumbnail of Single-domain antibody fragments with high conformational stability

Protein Science, 2009

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrare... more A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, ␤-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T m ‫ס‬ 60-80°C), and display high conformational stabilities (⌬G(H 2 O) ‫ס‬ 30-60 kJ mole −1 ). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications. Abbreviations: ANS, 8-anilino-1-naphtalene-sulfonic acid; BSA, bovine serum albumin; CD, circular dichroism; CDR, complementary determining region; csm, center of the spectral mass; Fab, Fv, scFv, and dsFv, antigenbinding fragment, variable fragment, single-chain variable fragment, and disulphide stabilized variable fragment of conventional antibodies, respectively; FTIR, Fourier transform infrared; GdmCl, guanidinium chloride; HEPES, N-(2-hydroxyethyl)piperazine-NЈ-2-ethanesulfonic acid; IPTG, isopropyl ␤-D-thiogalactopyranoside; IR, infrared; MOPS, 3-N-morpholinopropanosulfonic acid; RU, resonance units; SPR, surface plasmon resonance; V H , variable domain of immunoglobulin heavy chain; V L , variable domain of immunoglobulin light chain; V H H, variable domain of camelid heavy-chain antibody.

Research paper thumbnail of Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the α/β-barred packing)

"Protein Engineering, Design and Selection", 1991

We present here a spectroscopic structural characterization of octarellin, a recently reported de... more We present here a spectroscopic structural characterization of octarellin, a recently reported de novo protein modelled on alpha/beta-barrel proteins [K. Goraj, A. Renard and J.A. Martial (1990) Protein Engng, 3, 259-266]. Infrared and Raman spectra analyses of octarellin's secondary structure reveal the expected percentage of alpha-helices (30%) and a higher beta-sheet content (40%) than predicted from the design. When the Raman spectra obtained with octarellin and native triosephosphate isomerase (a natural alpha/beta-barrel) are compared, similar percentages of secondary structures are found. Thermal denaturation of octarellin monitored by CD confirms that its secondary structures are quite stable, whereas its native-like tertiary fold is not. Tyrosine residues, predicted to be partially hidden from solvent, are actually exposed as revealed by Raman and UV absorption spectra. We conclude that the attempted alpha/beta-barrel conformation in octarellin may be loosely packed. The criteria used to design octarellin are discussed and improvements suggested.

Research paper thumbnail of Pressure Effect on the Hydration Properties of Poly( N -isopropylacrylamide) in Aqueous Solution Studied by FTIR Spectroscopy

Macromolecules, 2005

ABSTRACT The hydration properties of poly(N-isopropylacrylamide) in aqueous solution were investi... more ABSTRACT The hydration properties of poly(N-isopropylacrylamide) in aqueous solution were investigated by Fourier transform infrared spectroscopy as a function of high hydrostatic pressure and compared to the thermally induced changes. We show that although both pressure and temperature induce a phase separation the underlying mechanisms are fundamentally different. It is well documented that increasing the temperature above the lower critical solution temperature causes a dehydration of the hydrophilic and hydrophobic moieties. By contrast, high pressure enhances the hydration of the hydrophilic amide group. Moreover, pressure strengthens the weak C−H···O hydrogen bonds between the hydrophobic alkyl groups and water, although a reorganization of the water network around the hydrophobic groups occurs during the phase separation. From this it is concluded that PNiPA remains in a coillike state at high pressure. In addition, we suggest that PNiPA is a good model for the study of the hydration properties of proteins.

Research paper thumbnail of Pressure-Induced Protein Unfolding in the Ternary System AOT−Octane−Water Is Different from that in Bulk Water

Langmuir, 2005

In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can i... more In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of R-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, R-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation.

Research paper thumbnail of Pressure-induced amorphization in biopolymers

Journal of Physics: Condensed Matter, 2002

Pressure-induced unfolding of proteins in solution shows analogies to the pressure-induced amorph... more Pressure-induced unfolding of proteins in solution shows analogies to the pressure-induced amorphization observed in some inorganic and polymer systems. More specifically, pressure gives rise to conformations that show a strong tendency to form supramolecular aggregates that have some relevance to molecular diseases. Hydrostatic pressure can tune the conformation of the intermediates along the unfolding/aggregation pathway. Pressure can also be used to probe the stability of the aggregate, and thus the interactions that are responsible for it. In particular, we demonstrate that pressure might be an interesting tool to study the fibril formation. Fourier transform infrared spectroscopy reveals the presence of fibril secondary structures other than random coil and intermolecular β-sheet.

Research paper thumbnail of Protein folding, unfolding and aggregation. Pressure induced intermediate states on the refolding pathway of horseradish peroxidase

Journal of Physics: Condensed Matter, 2004

We studied the refolding and aggregation of pressure unfolded proteins. Horseradish peroxidase wa... more We studied the refolding and aggregation of pressure unfolded proteins. Horseradish peroxidase was found to be very stable and no partially folded intermediates were populated during the refolding. However, the removal of the haem group or the Ca2+ ions or reduction of the disulfide bridge destabilized the protein, resulting in a significant amount of aggregation prone intermediate conformation. Substitution of

Research paper thumbnail of General discussion

Faraday Discussions of The Chemical Society, 1982

Research paper thumbnail of Effect of high pressure on the heat activation in vivo of trehalase in the spores of Phycomyces blakesleeanus

European journal of biochemistry / FEBS, 1980

The effect of pressure on the heat activation in vivo of trehalase in the spores of Phycomyces bl... more The effect of pressure on the heat activation in vivo of trehalase in the spores of Phycomyces blakesleeanus has been investigated in order to obtain information about the molecular mechanism of the activation. For a protein conformational change directly induced in the enzyme by the heat treatment an upward shift with about 2-6 K/1000 atm (1.013 X 10(5) kPa) is to be expected in the moderate high-pressure region. On the other hand, for a phospholipid phase transition causing the activation, a continuous upward shift with about 20 K/1000 atm is to be expected. For trehalase activation we find a continuous upward shift of the activation temperature with about 5-9 K/1000 atm. The denaturation of trehalase, which occurs at slightly higher temperatures, is influenced by pressure completely as expected for a protein conformational change. The application of high pressure during spore heat activation makes it possibe to break the dormancy of the spores without concomitant activation of tr...

Research paper thumbnail of Piezotropic unfolding of lysozyme in pure D2O at the outer edge of excess hydration

This Letter reports a FTIR study on the pressure-induced unfolding of lysozyme in pure D 2 O clos... more This Letter reports a FTIR study on the pressure-induced unfolding of lysozyme in pure D 2 O close to the conditions of non excess hydration. An essential population of the proteins apparently refold into its native structure after pressure release. It could furthermore be shown that down to a hydration of h = 2 (h = m prot /m D 2 O ; m = mass), the unfolding pressure did not vary with hydration. Hydration dependent behaviour was found with respect to the change of the wavenumbers of the a-helical structure during unfolding. This result is discussed with respect to the reversibility of unfolding in pure solvents and with the effects of hydrogen exchange.

Research paper thumbnail of Pressure-Temperature Effects on Proteins: Unfolding, Aggregation and Fibrillogenesis

Defect and Diffusion Forum, 2002

Research paper thumbnail of Two-dimensional Fourier-transform infrared correlation spectroscopy study of the high-pressure tuning of proteins

Vibrational Spectroscopy, 2000

Generalized two-dimensional (2D) correlation spectroscopy and evolving factor analysis (EFA) have... more Generalized two-dimensional (2D) correlation spectroscopy and evolving factor analysis (EFA) have been applied to the study of cationic curing reaction of mixtures of diglycidyl ether of bisphenol A (DGEBA) and c-valerolactone (c-VL). The reaction has been monitored by Fourier transform infrared spectroscopy. The reaction periods in which a chemical change takes place are identified using EFA and then the sequence of changes are established by means of the interpretation of the synchronous and asynchronous spectra obtained with 2D correlation spectroscopy. By combining this information, in the curing process, four reactions have been detected:

Research paper thumbnail of 2D FT-IR spectroscopy analysis of the pressure-induced changes in proteins

Vibrational Spectroscopy, 1999

In this paper, we apply for the first time two-dimensional (2D) correlation spectroscopy to analy... more In this paper, we apply for the first time two-dimensional (2D) correlation spectroscopy to analyze pressure-induced changes in proteins. We show that it is possible to distinguish between hydrogen-deuterium (H/D) exchange and conformational changes from the synchronous and the asynchronous spectra. From the sequence of the spectral changes it can be concluded that the initial partial unfolding of the secondary

Research paper thumbnail of Compressibility of the Heme Pocket of Substrate Analogue Complexes of Cytochrome P -450cam-CO. The Effect of Hydrostatic Pressure on the Soret Band

European Journal of Biochemistry, 1995

The effect of hydrostatic pressure on the electronic absorption spectrum of the carbon monoxide c... more The effect of hydrostatic pressure on the electronic absorption spectrum of the carbon monoxide complex of cytochrome P-450cam (CYP101) in the presence of various substrates was studied.

Research paper thumbnail of Pressure-Tuning the Conformation of Bovine Pancreatic Trypsin Inhibitor Studied by Fourier-Transform Infrared Spectroscopy

European Journal of Biochemistry, 1996

A hydrostatic pressure of 1 .5 GPa induces changes in the secondary structure of bovine pancreati... more A hydrostatic pressure of 1 .5 GPa induces changes in the secondary structure of bovine pancreatic tryspin inhibitor (BPTI) as revealed by the analysis of the amide I' band with Fourier-transform infrared (FTIR) spectroscopy in the diamond anvil cell. The features of the secondary structure remain distinct at high pressure suggesting that the protein does not unfold. The fitted percentages of the secondary structure elements during compression and decompression strongly suggest that the pressure-induced changes are reversible. The pressure-induced changes in the tyrosine side chain band are also reversible. The results demonstrate that the infrared technique explores different aspects of the behaviour of proteins in comparison with two published molecular dynamics studies performed up to 1

Research paper thumbnail of Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin

European Journal of Biochemistry, 1999

Research paper thumbnail of Effect of High Pressure, Detergents and Phaospholipase on the Break in the Arrhenius Plot of Azotobacter Nitrogenase

European Journal of Biochemistry, 1978

It is shown that lipids are responsible for the breaks in the Arrhenius plots of Azotohuctr~r nit... more It is shown that lipids are responsible for the breaks in the Arrhenius plots of Azotohuctr~r nitrogenase. The physical evidence is that temperature at which the break occurs incrcases with increasing pressure by 20 KjlOOO atm. This is in agreement with the pressurc dependence of the transition temperature of several synthetic phospholipids. We also find the same pressure dependence for the broad transitions observed in Eschericlzia coli phosphatidylethanolamine and in the membrane lipids from Azotobucter itself. Detergents and phospholipase remove the break. Reconstruction can be performed only with specific phospholipids.

Research paper thumbnail of Intermolecular Interactions of Proteins under Pressure. Aggregation, Dissociation, Chaperoning

Defect and Diffusion Forum, 2002

Research paper thumbnail of Molecular mechanisms of pressure induced conformational changes in BPTI

Proteins: Structure, Function, and Genetics, 1996

Skip to Main Content. ...

Research paper thumbnail of Extreme heat‐ and pressure‐resistant 7‐kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single‐point amino acid substitution

Proteins: Structure, Function, and Genetics, 1997

This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfa... more This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability. As observed in the NMR, FTIR, and CD spectra, wild-type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar. The pressure-induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented. These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance.

Research paper thumbnail of High pressure effects on protein structure and function

Proteins: Structure, Function, and Genetics, 1996

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and... more Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity. 0 1996 Wiley-Liss, Inc.

Research paper thumbnail of Single-domain antibody fragments with high conformational stability

Protein Science, 2009

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrare... more A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, ␤-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T m ‫ס‬ 60-80°C), and display high conformational stabilities (⌬G(H 2 O) ‫ס‬ 30-60 kJ mole −1 ). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications. Abbreviations: ANS, 8-anilino-1-naphtalene-sulfonic acid; BSA, bovine serum albumin; CD, circular dichroism; CDR, complementary determining region; csm, center of the spectral mass; Fab, Fv, scFv, and dsFv, antigenbinding fragment, variable fragment, single-chain variable fragment, and disulphide stabilized variable fragment of conventional antibodies, respectively; FTIR, Fourier transform infrared; GdmCl, guanidinium chloride; HEPES, N-(2-hydroxyethyl)piperazine-NЈ-2-ethanesulfonic acid; IPTG, isopropyl ␤-D-thiogalactopyranoside; IR, infrared; MOPS, 3-N-morpholinopropanosulfonic acid; RU, resonance units; SPR, surface plasmon resonance; V H , variable domain of immunoglobulin heavy chain; V L , variable domain of immunoglobulin light chain; V H H, variable domain of camelid heavy-chain antibody.

Research paper thumbnail of Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the α/β-barred packing)

"Protein Engineering, Design and Selection", 1991

We present here a spectroscopic structural characterization of octarellin, a recently reported de... more We present here a spectroscopic structural characterization of octarellin, a recently reported de novo protein modelled on alpha/beta-barrel proteins [K. Goraj, A. Renard and J.A. Martial (1990) Protein Engng, 3, 259-266]. Infrared and Raman spectra analyses of octarellin's secondary structure reveal the expected percentage of alpha-helices (30%) and a higher beta-sheet content (40%) than predicted from the design. When the Raman spectra obtained with octarellin and native triosephosphate isomerase (a natural alpha/beta-barrel) are compared, similar percentages of secondary structures are found. Thermal denaturation of octarellin monitored by CD confirms that its secondary structures are quite stable, whereas its native-like tertiary fold is not. Tyrosine residues, predicted to be partially hidden from solvent, are actually exposed as revealed by Raman and UV absorption spectra. We conclude that the attempted alpha/beta-barrel conformation in octarellin may be loosely packed. The criteria used to design octarellin are discussed and improvements suggested.

Research paper thumbnail of Pressure Effect on the Hydration Properties of Poly( N -isopropylacrylamide) in Aqueous Solution Studied by FTIR Spectroscopy

Macromolecules, 2005

ABSTRACT The hydration properties of poly(N-isopropylacrylamide) in aqueous solution were investi... more ABSTRACT The hydration properties of poly(N-isopropylacrylamide) in aqueous solution were investigated by Fourier transform infrared spectroscopy as a function of high hydrostatic pressure and compared to the thermally induced changes. We show that although both pressure and temperature induce a phase separation the underlying mechanisms are fundamentally different. It is well documented that increasing the temperature above the lower critical solution temperature causes a dehydration of the hydrophilic and hydrophobic moieties. By contrast, high pressure enhances the hydration of the hydrophilic amide group. Moreover, pressure strengthens the weak C−H···O hydrogen bonds between the hydrophobic alkyl groups and water, although a reorganization of the water network around the hydrophobic groups occurs during the phase separation. From this it is concluded that PNiPA remains in a coillike state at high pressure. In addition, we suggest that PNiPA is a good model for the study of the hydration properties of proteins.

Research paper thumbnail of Pressure-Induced Protein Unfolding in the Ternary System AOT−Octane−Water Is Different from that in Bulk Water

Langmuir, 2005

In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can i... more In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of R-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, R-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation.

Research paper thumbnail of Pressure-induced amorphization in biopolymers

Journal of Physics: Condensed Matter, 2002

Pressure-induced unfolding of proteins in solution shows analogies to the pressure-induced amorph... more Pressure-induced unfolding of proteins in solution shows analogies to the pressure-induced amorphization observed in some inorganic and polymer systems. More specifically, pressure gives rise to conformations that show a strong tendency to form supramolecular aggregates that have some relevance to molecular diseases. Hydrostatic pressure can tune the conformation of the intermediates along the unfolding/aggregation pathway. Pressure can also be used to probe the stability of the aggregate, and thus the interactions that are responsible for it. In particular, we demonstrate that pressure might be an interesting tool to study the fibril formation. Fourier transform infrared spectroscopy reveals the presence of fibril secondary structures other than random coil and intermolecular β-sheet.

Research paper thumbnail of Protein folding, unfolding and aggregation. Pressure induced intermediate states on the refolding pathway of horseradish peroxidase

Journal of Physics: Condensed Matter, 2004

We studied the refolding and aggregation of pressure unfolded proteins. Horseradish peroxidase wa... more We studied the refolding and aggregation of pressure unfolded proteins. Horseradish peroxidase was found to be very stable and no partially folded intermediates were populated during the refolding. However, the removal of the haem group or the Ca2+ ions or reduction of the disulfide bridge destabilized the protein, resulting in a significant amount of aggregation prone intermediate conformation. Substitution of