Papers by Michael W. Lassalle
Artificial selection, a method by which evolution occurs, is a process in which an organism is mo... more Artificial selection, a method by which evolution occurs, is a process in which an organism is modified to fulfill a specific purpose. For instance, the evolution of corn dates back about 10,000 years ago. Farmers in Mexico recognized that not all plants were identical and that some were locally more adapted. Through unconscious selection and open pollination, the first landraces developed. Further progresses allowed for conscious selection. However, farmers and companies quickly realized that crossing parent plants to create hybrids was too time-consuming to be economically viable. Backcrossing reduced the time required to obtain an organism with the desired trait. Further technological developments made organic food possible through the utilization of atomic gardening. Recent progress in genetics has enabled creation of so-called GMOs, or genetically modified organisms. All of the developed methods (open pollination, mutation breeding, atomic farming, CRISPR/Cas) have a common goal: to adjust the organism to express a specific trait. Nevertheless, some of the methods are seen as potentially dangerous. Furthermore, the scientists' and public opinion on GMOs are different which raise concerns about scientific and critical literacy regarding GMOs. The present article investigates the misconception that distinguish genetically modified organisms based on the method by which they have been created and relates this misconception to literacy (scientific/critical) and critical thinking. A new term, " Adjusted Organism, " is proposed to enable a fresh, unbiased view for future discussions.
Protein Expression and Purification, 2013
Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC)... more Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein complex should be studied with assembled hcTn rather than a short fragment of a subunit or the subunit itself. Here, we describe the expression and assembly of active hcTn in Escherichia coli, a novel method that is rapid and simple, and produces large amounts of functional hcTn.
Bioscience, Biotechnology, and Biochemistry, 2010
Mutations in Troponin I (TnI) and Troponin T (TnT) are closely linked to familial hypertrophic ca... more Mutations in Troponin I (TnI) and Troponin T (TnT) are closely linked to familial hypertrophic cardiomyopathy (FHC) and hypertrophic cardiomyopathy (HCM), but the underlying molecular mechanism is not yet well understood. There might be a close link between the defective dynamic properties and the functional aberrations of hcTroponin. To prove this hypothesis, we undertook detailed NMR relaxation measurements of [(2)H, (13)C, (15)N] labeled proteins reconstituted into hcTroponin in both the Ca(2+)- and the Mg(2+)-loaded state. The wild-type TnI and two FHC causing mutations, TnI(G203S) and TnI(DeltaK183), were investigated. To ensure that defective dynamic properties are not only a particular feature for mutations in the flexible part of TnI, measurements of the TnT mutation TnT(R278P) were also performed. For all mutations significant dynamic changes in the area for Troponin C (TnC) and actin-Tm binding were obtained. These measurements provide important information to understand the functional aberrations of FHC and HCM causing mutation in human cardiac Troponin (hcTn).
J Mol Biol, 1998
The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison ... more The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-α-helix motif of the wild-type structure is given up. Instead a homotetrameric four-α-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71°C characteristic of the wild-type protein to 101°C for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer)−1 to 195.1 kJ (mol of tetramer)−1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of ΔH=1073±30 kJ (mol of tetramer)−1 and a heat capacity change at the transition temperature of ΔNDCp=14.9(±)3% kJ (mol of tetramer × K)−1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van’t Hoff enthalpy values.
J Mol Biol, 2000
The thermodynamic stability of staphylococcal nuclease was studied against the variation of both ... more The thermodynamic stability of staphylococcal nuclease was studied against the variation of both temperature and pressure by utilizing 1H NMR spectroscopy at 750 MHz in 20 mM Mes buffer containing 99.9 % 2H2O, pH 5.3. Equilibrium fractions of folded and unfolded protein species were evaluated with the proton signals of two histidine residues as monitor in the pressure range of 30-3300 bar and in the temperature range of 1.5 °C-35 °C. From the multi-parameter fit of the experimental data to the Gibbs energy equation expressed as a simultaneous function of pressure and temperature, we determined the compressibility change (Δβ), the volume change at 1 bar (ΔV°) and the expansivity change (Δα) upon unfolding among other thermodynamic parameters: Δβ=0.02(±0.003) ml mol−1 bar−1; Δα=1.33(±0.2) ml mol−1 K−1; ΔV°=−41.9(±6.3) ml mol−1 (at 24 °C); ΔG°=13.18(±2) kJ mol−1 (at 24 °C); ΔCp=13.12(±2) kJ mol−1 K−1; ΔS°=0.32(±0.05) kJ mol−1 K−1 (at 24 °C). The result yields a three-dimensional free energy surface, i.e. the free energy-landscape of staphylococcal nuclease on the P-T plane. The significantly positive Δβ and Δα values suggest that, in the pressure-denatured state, staphylococcal nuclease forms a loosely packed and fluctuating structure. The slight but statistically significant difference between the unfolding transitions of the His8 and His124 environments is considered to reflect local fluctuations in the native state, leading to pre-melting of the His124 environment prior to the cooperative unfolding of the major part of the protein.
Methods in Molecular Biology
Recent development of high-pressure cells for a variety of spectroscopic methods has enabled the ... more Recent development of high-pressure cells for a variety of spectroscopic methods has enabled the use of pressure as one of the commonly used perturbations along with temperature and chemical perturbations to study folding/unfolding reactions of proteins. Although various high-pressure spectroscopy techniques have their own significance, high-pressure nuclear magnetic resonance (NMR) is unique in that it allows one to gain residue-specific and atom-detailed information from proteins under pressure. Furthermore, because of a peculiar volume property of a protein, high-pressure NMR allows one to obtain structural information of a protein in a wide conformational space from the bottom to the upper region of the folding funnel, giving structural reality for the "open" state of a protein proposed from hydrogen exchange. The method allows a link between equilibrium folding intermediates and the kinetic intermediates, and manifests a new view of proteins as dynamic entities amply fluctuating among the folded, intermediate, and unfolded sub ensembles. This chapter briefly summarizes the technique, the principle, and the ways to use high-pressure NMR for studying protein folding.
Methods in molecular biology (Clifton, N.J.), 2007
Recent development of high-pressure cells for a variety of spectroscopic methods has enabled the ... more Recent development of high-pressure cells for a variety of spectroscopic methods has enabled the use of pressure as one of the commonly used perturbations along with temperature and chemical perturbations to study folding/unfolding reactions of proteins. Although various high-pressure spectroscopy techniques have their own significance, high-pressure nuclear magnetic resonance (NMR) is unique in that it allows one to gain residue-specific and atom-detailed information from proteins under pressure. Furthermore, because of a peculiar volume property of a protein, high-pressure NMR allows one to obtain structural information of a protein in a wide conformational space from the bottom to the upper region of the folding funnel, giving structural reality for the "open" state of a protein proposed from hydrogen exchange. The method allows a link between equilibrium folding intermediates and the kinetic intermediates, and manifests a new view of proteins as dynamic entities amply ...
Proteins: Structure, Function, and Genetics, 2001
Cavities or packing defects in proteins may generally be related with the dynamics and function o... more Cavities or packing defects in proteins may generally be related with the dynamics and function of a protein. In the c-Myb R2 subdomain, its single cavity has been shown to be crucial for its DNA recognition. Cavities are also considered important in determining the pressure stability of a protein. In the present work, high-pressure proton nuclear magnetic resonance ((1)H NMR) spectroscopy at 750 MHz is used to study the effect of a cavity-filling mutation (V103L) on the stability of the c-Myb R2 subdomain in the pressure range between 1 and 3,700 bar at 5 degrees C. A dramatic increase in the pressure stability of the c-Myb R2 subdomain is attained, from which we estimate the cavity size to be 35.3 A(3), in good agreement with literature values. We also evaluated the increase in thermodynamic stability DeltaG(0)(1bar) from 5.35 kJ/mol to 7.34 kJ/mol by the mutation, giving a clear example of the effect of a cavity on the global stability of a globular protein.
Protein Science, 2003
Pressure-induced unfolding of a molten globule (MG) was studied in a residue-specific manner with... more Pressure-induced unfolding of a molten globule (MG) was studied in a residue-specific manner with 1 H-15 N two-dimensional NMR spectroscopy using a variant of human ␣-lactalbumin (␣-LA), in which all eight cysteines had been replaced with alanines (all-Ala ␣-LA). The NMR spectrum underwent a series of changes from 30 to 2000 bar at 20°C and from −18°C to 36°C at 2000 bar, showing a highly heterogeneous unfolding pattern according to the secondary structural elements of the native structure. Unfolding began in the loop part of the -domain, and then extended to the remainder of the -domain, after which the ␣-domain began to unfold. Within the ␣-domain, the pressure stability decreased in the order: D-helix ∼ 3 10 -helix > Chelix ∼ B-helix > A-helix. The D-helix, C-terminal 3 10 -helix and a large part of B-and C-helices did not unfold at 2000 bar, even at 36°C or at −18°C. The results verify that the MG state consists of a mixture of variously unfolded conformers from the mostly folded to the nearly totally unfolded that differ in stability and partial molar volume. Not only heat but also cold denaturation was observed, supporting the view that the MG state is stabilized by hydrophobic interactions.
Pigment Cell Research, 2003
Melanin pigments produced in human melanocytes are classified into two categories; black coloured... more Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish-yellow pheomelanin. Stimulation of melanocytes with a-melanocyte-stimulating hormone (a-MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis-stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of a-MSH. The amounts of eumelanin and pheomelanin were quantified using high-performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of a-MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with a-MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of a-MSH, may contribute to UV-induced hyperpigmentation by enhancing eumelanogenesis.
Journal of Molecular Biology, 1998
The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison ... more The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the ®ve amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-a-helix motif of the wild-type structure is given up. Instead a homotetrameric four-a-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71 C characteristic of the wild-type protein to 101 C for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer) À1 to 195.1 kJ (mol of tetramer) À1 . The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of ÁH 1073 AE 30 kJ (mol of tetramer) À1 and a heat capacity change at the transition temperature of Á D N C p 14.9(AE)3% kJ (mol of tetramer  K) À1 . The two-state nature of the unfolding reaction is re¯ected in coinciding calorimetric and van't Hoff enthalpy values.
Journal of Molecular Biology, 2000
The thermodynamic stability of staphylococcal nuclease was studied against the variation of both ... more The thermodynamic stability of staphylococcal nuclease was studied against the variation of both temperature and pressure by utilizing 1 H NMR spectroscopy at 750 MHz in 20 mM Mes buffer containing 99.9 % 2 H 2 O, pH 5.3. Equilibrium fractions of folded and unfolded protein species were evaluated with the proton signals of two histidine residues as monitor in the pressure range of 30-3300 bar and in the temperature range of 1.5 C-35 C. From the multi-parameter ®t of the experimental data to the Gibbs energy equation expressed as a simultaneous function of pressure and temperature, we determined the compressibility change (Áb), the volume change at 1 bar (ÁV ) and the expansivity change (Áa) upon unfolding among other thermodynamic parameters: Áb 0.02(AE0.003) ml mol À1 bar À1 ; Áa 1.33(AE0.2) ml mol À1 K À1 ; ÁV À 41.9(AE6.3) ml mol À1 (at 24 C); ÁG 13.18(AE2) kJ mol À1 (at 24 C); ÁC p 13.12(AE2) kJ mol À1 K À1 ; ÁS 0.32(AE0.05) kJ mol À1 K À1 (at 24 C). The result yields a three-dimensional free energy surface, i.e. the free energy-landscape of staphylococcal nuclease on the P-T plane. The signi®cantly positive Áb and Áa values suggest that, in the pressure-denatured state, staphylococcal nuclease forms a loosely packed and¯uctuating structure. The slight but statistically signi®cant difference between the unfolding transitions of the His8 and His124 environments is considered to re¯ect local¯uctuations in the native state, leading to pre-melting of the His124 environment prior to the cooperative unfolding of the major part of the protein.
Journal of Bioscience and Bioengineering, 2011
The wheat germ cell-free system is composed out of five basic steps, growth of Escherichia coli h... more The wheat germ cell-free system is composed out of five basic steps, growth of Escherichia coli harboring plasmid, first colony-PCR, second PCR, transcription, and translation. Improvements of culture medium, colony based PCR, and modifications within the split primer set of the second PCR amplify both DNA and RNA levels. This yields more than 5 times increase in protein amount for pEU-originated templates. Especially, for the low PCR-amplifiable vectors with pETorigin, it leads to 30 fold higher product amount in translation. This broadens the range of usable vectors, overcoming the existing cell-free system limitations for high-throughput protein screening. Noteworthily, the system successfully maintains translation by S-30 cell-free extract below 30 OD. In conclusion, this improved firm cell-free system reduces cost and enables robotic automation and high-throughput thermodynamic analysis, especially for proteins that are difficult to be expressed.
Biological Chemistry, 2000
Previous DSC and X-ray studies on RM6, a loop deletion mutant of wtROP protein, have shown that r... more Previous DSC and X-ray studies on RM6, a loop deletion mutant of wtROP protein, have shown that removal of five amino acids from the loop causes a dramatic reorganization of the wild-type structure. The new tetrameric molecule exhibits a significantly higher stability (Lassalle, M.W. et al., J. Mol. Biol., 1998, 279, 987-1000) and unfolds in a second order reaction (Lassalle, M.W. and Hinz, H.-J., Biochemistry, 1998, 37, 8465-8472). In the present investigation we report extensive refolding studies of RM6 at different temperatures and GdnHCl concentrations monitored by CD and fluorescence to probe for changes in secondary and tertiary structure, respectively. The measurements permitted us to determine activation parameters as a function of denaturant concentration. The results demonstrate convincingly that the variation with GdnHCl concentration of the activation parameters deltaH#, deltaS# and deltaG# is very similar for unfolding and refolding. For both processes the activation properties approach a maximum in the vicinity of the denaturant concentration, c(K=1), where the equilibrium constant equals 1, i.e. deltaG0 equals zero. CD and fluorescence refolding kinetics are described by identical constants suggesting that the formation of secondary and tertiary structure occurs simultaneously. Refolding is, however, characterized by a more complex mechanism than unfolding. Although the general pattern is dominated by the sequence monomers to dimers to tetramers, parallel side reactions involving dimers and monomers have to be envisaged in the initial folding phase, supporting the view that the native state of RM6 can be reached by several rather than a single pathway.
Biochemistry, 1998
Comprehensive kinetic studies were carried out on the unfolding properties of RM6 as a function o... more Comprehensive kinetic studies were carried out on the unfolding properties of RM6 as a function of GdnHCl concentration and temperature. This protein is a mutant resulting from the dimeric wild-type CoLE1-ROP protein by deletion of 5 amino acids (Asp 30, Ala 31, Asp 32, Glu 33, Gln 34) in the loop of each monomer. The deletion has dramatic consequences. The dimeric 4-R-helix structure characteristic of the wild-type protein is completely reorganized and the RM6 structure can be described as a tetrameric R helix of extended monomers without loops. These extraordinary structural changes are accompanied by an enormous increase in transition temperature from 71 to 101°C. These features have been discussed in a separate publication (1). The remarkable change in thermal stability of RM6 should be reflected in significant changes in the folding rate constants. This was observed in the present unfolding studies. Decay of tetrameric RM6 was monitored by circular dichroism (CD) and fluorescence to probe for changes in both secondary and tertiary structure, respectively. The identity of the kinetic parameters obtained from the two techniques supports the view that secondary and tertiary structure break down simultaneously. However, the most intriguing result is the finding that unfolding of tetrameric RM6 can be described very well by a second-order reaction. The magnitude of the second-order rate constant k 2 varies dramatically with both temperature and denaturant concentration. At 25°C and 6.5 M GdnHCl concentration k 2 is 4200 L‚(mol of dimer) -1 ‚s -1 , whereas at 4.4 M GdnHCl a value of k 2 ) 0.9 L‚(mol of dimer) -1 ‚s -1 is observed. Correspondingly, apparent activation enthalpies show a strong increase from ∆H # ) 29.1 kJ‚mol -1 at 6.5 M GdnHCl to ∆H # ) 79.7 kJ‚mol -1 at 4.4 M GdnHCl. A mechanism involving a dimeric intermediate is suggested which permits a consistent interpretation of the findings. † This research was supported by a scholarship from the Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie e.V. to M.W.L. and by grants from the European Community Bridge program and the DFG to H.-J.H.
Artificial selection, a method by which evolution occurs, is a process in which an organism is mo... more Artificial selection, a method by which evolution occurs, is a process in which an organism is modified to fulfill a specific purpose. For instance, the evolution of corn dates back about 10,000 years ago. Farmers in Mexico recognized that not all plants were identical and that some were locally more adapted. Through unconscious selection and open pollination, the first landraces developed. Further progresses allowed for conscious selection. However, farmers and companies quickly realized that crossing parent plants to create hybrids was too time-consuming to be economically viable. Backcrossing reduced the time required to obtain an organism with the desired trait. Further technological developments made organic food possible through the utilization of atomic gardening. Recent progress in genetics has enabled creation of so-called GMOs, or genetically modified organisms. All of the developed methods (open pollination, mutation breeding, atomic farming, CRISPR/Cas) have a common goal: to adjust the organism to express a specific trait. Nevertheless, some of the methods are seen as potentially dangerous. Furthermore, the scientists' and public opinion on GMOs are different which raise concerns about scientific and critical literacy regarding GMOs. The present article investigates the misconception that distinguish genetically modified organisms based on the method by which they have been created and relates this misconception to literacy (scientific/critical) and critical thinking. A new term, " Adjusted Organism, " is proposed to enable a fresh, unbiased view for future discussions.
Key-words- Genetically Modified Organisms, Critical literacy, Scientific literacy, Bioethics, Gene editing, GM food, Governance of science and technology
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Papers by Michael W. Lassalle
Key-words- Genetically Modified Organisms, Critical literacy, Scientific literacy, Bioethics, Gene editing, GM food, Governance of science and technology
Key-words- Genetically Modified Organisms, Critical literacy, Scientific literacy, Bioethics, Gene editing, GM food, Governance of science and technology