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K Heremans

Publications and source records attributed to K Heremans.

At least 19 recordsLinked to original sources
PubMed1998

Protein structure and dynamics at high pressure.

The effect of pressure on the structure and dynamics of proteins is discussed in the framework of the pressure-temperature stability phase diagram. The elastic (reversible) properties, thermal expansion, compressibility and heat capacity, are correlated with the entropy, volume, and the coupling between entropy and volume fluctuations respectively. The experimental approaches that can be used to measure these quantities are reviewed. The plastic (conformational) changes reflect the changes in these properties in the cold, pressure and heat denaturation.

Chemical Phenomena↗
PubMed1997

A prediction of DPP IV/CD26 domain structure from a physico-chemical investigation of dipeptidyl peptidase IV (CD26) from human seminal plasma.

Human DPP IV, isolated from seminal plasma by means of immobilised adenosine deaminase, occurs in different forms which are distinguishable by net charge and native molecular weight. Charge differences arise primarily from different degrees of glycosylation containing various amounts of sialic acid. The majority of DPP IV isolated from total seminal plasma consists of the extracellular part of the protein starting at Gly-31. It is a very stable protein resisting high concentrations of denaturant. Unfolding experiments under reducing conditions are indicative of the existence of at least two domains which function independently. One of these domains is highly stabilised by disulfide bonds. Disruption of the disulfide bonds does not affect the activity, the dimeric state nor the adenosine deaminase binding properties of the protein but renders it more susceptible to proteolysis. The low-angle X-ray scattering spectrum is consistent with a model for a protein containing two subunits, each composed of three domains linked by flexible regions with low average mass. The secondary structure composition, determined by FTIR spectrometry, indicates that 45% of the protein consists of beta-sheets, which is higher than expected from computed secondary structure predictions. Our results provide compelling experimental evidence for the three-domain structure of the extracellular part of DPP IV.

Amino Acid Sequence↗
PubMed1997

Extreme heat- and pressure-resistant 7-kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single-point amino acid substitution.

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.

Alanine↗
PubMed1996

Identification of local carboxy-terminal hydrophobic interactions essential for folding or stability of chloramphenicol acetyltransferase.

The role of the carboxy terminal in folding and stabilization of type I chloramphenicol acetyltransferase (CAT1) has been studied by mutagenesis and Fourier transform infrared analysis. We have shown that a CAT mutant truncated by seven amino acid residues folds into active protein. In this study, the last three residues of this truncated CAT mutant were randomized to detect structural information required for achieving a native enzyme conformation. Statistical analysis of sequencing data from randomly chosen mutants revealed that the amino-terminal CAT fragment of 212 amino acid residues is the shortest deletion mutant able to adopt a soluble, enzymatically active structure. This minimal length corresponds to a protein with full-length alpha5-helix in the three-dimensional crystal structure of CAT type III. The amino acid preferences at the carboxy terminal in the randomization experiments suggest that this helix also forms completely in the shortened CAT mutants. In addition correct folding and/or stabilization requires the formation of a hydrophobic + microdomain at the end of the alpha5-helix. The role of this hydrophobic interaction in CAT folding and structure stabilization is discussed.

Amino Acid Sequence↗
PubMed1996

Pressure-tuning the conformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy.

A hydrostatic pressure of 1.5 GPa induces changes in the secondary structure of bovine pancreatic trypsin 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 GPa [Kitchen, D.B., Reed, L.H. & Levy, R.M.(1992) Biochemistry 31, 10083-10093] and 500 MPa [Brunne, R.M. & van Gunsteren, W.F.(1993) FEBS Lett. 323, 215-217]. A possible explanation for the difference is the time scale of the experiments.

Amides↗
PubMed1996

High pressure effects on protein structure and function.

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.

Biophysics↗
PubMed1996

Molecular mechanisms of pressure induced conformational changes in BPTI.

We have performed a 800 ps molecular dynamics simulation of bovine pancreatic trypsin inhibitor (BPTI) in water coupled to a pressure bath at 1, 10,000, 15,000, and 20,000 bar. The simulation reproduces quite well the experimental behavior of the protein under high pressure. The protein keeps its globular form, but adopts a different conformation with a very small reduction in volume. Some residues in the hydrophobic core become exposed to water and a large part of the secondary structure of the protein, (60% of the sheet structure and 40% of the helical structure) is denatured between 10 and 15 kbar. This is in good agreement with experimental data (Goossens, K., et al. Eur. J. Biochem, 236:254-262, 1996) that show denaturation of BPTI between 8 and 14 kbar. A further increase of the pressure results in a freezing of the protein as deduced from the large decrease of the mobility of the residues. During the simulation, the normal structure of water changes from an ice Ih-like to an ice VI-like structure, while keeping the liquid state. The driving force of the high pressure induced conformational transition seems be the higher compressibility of the water compared with the protein. This produces a change in the solvent properties and leads to penetration of the solvent into the hydrophobic core.

Aprotinin↗
PubMed1995

Compressibility of the heme pocket of substrate analogue complexes of cytochrome P-450cam-CO. The effect of hydrostatic pressure on the Soret band.

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. With increasing pressure the wavenumber of the Soret band in the cytochrome P-450-CO complex shifts linearily to lower values (red-shift) and the half-width increases (broadening). The microscopic theory of solvent-solute interaction discussed by Laird and Skinner is used to explain the observed pressure effects. According to this theory, the slope of the red-shift of the Soret band is related to the compressibility of the chromophore environment, that is the heme moiety of the hemoproteins. It was found that the slope of the red-shift and the slope of the broadening of the Soret band for the CO complex in the presence of various substrate analogues increase with the decrease of the initial high-spin content at 0.1 MPa in the oxidized state. Variation of the high-spin content reflects the changes in the number of water molecules and/or changes in the polarity of the heme environment. The higher compressibility of the cytochrome P-450 complexes with the substrate analogues, which induce a lower degree of the high-spin content in the oxidized protein, is explained by the ability of the water molecules in the heme moiety to transmit the pressure effect on the protein structure to the heme chromophore. Therefore, a larger pressure-induced red-shift of the Soret band in the CO complex of cytochrome P-450cam might indicate a higher water content in the heme environment.

Camphor 5-Monooxygenase↗
PubMed1995

Pressure effects on the stability of lipoxygenase: Fourier transform-infrared spectroscopy (FT-IR) and enzyme activity studies.

Fourier transform infrared spectroscopy (FT-IR) studies of lipoxygenase at pressures of up to 1.2 GPa have shown changes in the amide I' band which correlate to structural changes of the enzyme. The shift of the frequency maximum of the amide I' band at about 600 MPa suggests a cooperative change in the secondary structure of the protein. Studies of the changes in band width have shown the structural changes at 600 MPa to be irreversible. This has been confirmed by studies of enzyme activity after pressure treatment: exposure to 600 MPa for 30 min (40 degrees C) clearly reduced the activity of lipoxygenase. Anodic gel electrophoresis under non-denaturating conditions revealed a decrease in native protein parallel to the activity loss. A pressure-temperature-phase diagram for soybean lipoxygenase was established.

Enzyme Stability↗
PubMed1992

Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out. The secondary structure of the molecule is composed of 54% beta-sheet with less than 5% ordered helix. The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted. The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8. Two proteinase-resistant mutants of PDGF [( Ser28]- and [Pro32]-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB. These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB.

Mutagenesis, Site-Directed↗
PubMed1991

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the alpha/beta-barrel packing).

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.

Circular Dichroism↗
PubMed1989

Raman spectroscopic study of the changes in secondary structure of chymotrypsin: effect of pH and pressure on the salt bridge.

Conformational changes of alpha-chymotrypsin, induced by pH and pressure, have been studied with Raman spectroscopy. The secondary structure of alpha-chymotrypsin, chymotrypsinogen and DFP-chymotrypsin has been calculated by a singular value analysis of the Raman amide-I band. The changes in secondary structure, with pH and pressure titration of alpha-chymotrypsin, indicate a conformational transition. The salt bridge between Asp-194 and Ile-16 is disrupted, and the enzyme becomes inactive. No changes are observed for chymotrypsinogen. It is concluded that the proenzyme exhibits the same conformation at different pH values as alpha-chymotrypsin at alkaline pH. The results for DFP-chymotrypsin indicate that the active conformation is stabilized by the presence of the DFP inhibitor in the binding site.

Chymotrypsin↗
PubMed1989

Secondary structure analysis of tubulin and microtubules with Raman spectroscopy.

Raman spectroscopy is used to study the secondary structure of tubulin in the assembled and the dissociated states from the analysis of the amide-I band. Essentially two states are recognized: the GTP- and the GDP-bound state, differing in alpha-helix and antiparallel beta-sheet content. Microtubules give a spectrum which is very similar to the GDP-bound state. MAPs and temperature have minor effects, while increasing the pH up to 8 causes a reduction in alpha-helix content and a increase in antiparallel beta-sheet. The binding of demecolcine also induces structural changes which are similar to the GDP-bound state.

Amides↗
PubMed1988

Pressure effects on protein secondary structure and hydrogen deuterium exchange in chymotrypsinogen: a Fourier transform infrared spectroscopic study.

Infrared spectra of chymotrypsinogen in 2H2O have been measured as a function of pressure up to 30 kbar. An irreversible denaturation is induced at 7.6 kbar where the contributions of the random coil and turn conformational substructures to the protein structure increase dramatically at the expense of the contributions of the alpha-helix and beta-sheet substructures. The pressure at which denaturation starts is higher when pressure is applied more slowly. The rate of H-2H exchange of the interior labile protons on the amide groups is dramatically increased by external pressure and is closely associated with the global conformational structure of the protein. This suggest that pressure denaturation involves the bulk of the molecule. The results are compared with those obtained from other experimental approaches and with temperature-induced denaturation.

Amides↗
PubMed1988

Pressure-induced effects on cytochrome oxidase: the aerobic steady state.

If cytochrome c oxidase is subjected to pressure during the aerobic steady state, large spectral changes are apparent. These seem to be associated with the inhibition of electron transport within the oxidase. The volume change for the transition is about 80 mL/mol. When the oxidase in the aerobic steady state, with porphyrin cytochrome c (the iron-free derivative of cytochrome c) bound to it, is subjected to pressure, the porphyrin derivative is released. This results from a change in the dissociation constant of the complex. Whereas the dissociation constant during turnover is about 1.25 X 10(-8) M, during pressure-induced inhibition the dissociation constant appears to be about an order of magnitude greater. It appears as though the binding site of the inhibited, partially reduced enzyme more closely resembles that of the fully reduced enzyme than that of the enzyme during the aerobic steady state.

Aerobiosis↗
PubMed1983

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase.

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.

Chloride Peroxidase↗
PubMed1982

Activation volumes for horseradish peroxidase compound II reactions.

The activation volumes for the reactions of horseradish peroxidase compound II with L-tyrosine, 3-iodo-L-tyrosine, p-aminobenzoic acid and ferrocyanide were determined by using a high-pressure stopped-flow technique at 25 degrees C and pH 7. For the tyrosines, the solvent electrostriction accompanying substrate ionization and H+ transfer from the substituted phenol to a basic group of the enzyme can account for the observed negative activation volumes. For p-aminobenzoic acid a simple electron transfer without H+ transfer appears to occur. The positive activation volume for ferrocyanide may be explained in terms of electron transfer associated with a large change in electrostriction of the inorganic redox couple.

Enzyme Activation↗