Jochen Balbach | Martin Luther University Halle-Wittenberg (original) (raw)
Papers by Jochen Balbach
PLOS ONE, 2015
The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members se... more The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.
Nature Structural Biology, 1995
Proceedings of the National Academy of Sciences, 1997
Protein folding can be described in terms of the development of specific contacts between residue... more Protein folding can be described in terms of the development of specific contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with ␣-lactalbumin reveal the existence of extensive NOEs between aromatic and aliphatic protons in the archetypal molten globule formed by this protein at low pH. Analysis of their time development provides direct evidence for near-native compactness of this state. Through a rapid refolding procedure the NOE intensity can be transferred efficiently into the resolved and assigned spectrum of the native state. This demonstrates the viability of using this approach to map out time-averaged interactions between residues in a partially folded protein.
Proceedings of the National Academy of Sciences, 2008
Repeat proteins are widespread in nature, with many of them functioning as binding molecules in p... more Repeat proteins are widespread in nature, with many of them functioning as binding molecules in protein-protein recognition. Their simple structural architecture is used in biotechnology for generating proteins with high affinities to target proteins. Recent folding studies of ankyrin repeat (AR) proteins revealed a new mechanism of protein folding. The formation of an intermediate state is rate limiting in the folding reaction, suggesting a scaffold function of this transient state for intrinsically less stable ARs. To investigate a possible common mechanism of AR folding, we studied the structure and folding of a new thermophilic AR protein (tANK) identified in the archaeon Thermoplasma volcanium. The x-ray structure of the evolutionary much older tANK revealed high homology to the human CDK inhibitor p19 INK4d , whose sequence was used for homology search. As for p19 INK4d , equilibrium and kinetic folding analyses classify tANK to the family of sequential three-state folding proteins, with an unusual fast equilibrium between native and intermediate state. Under equilibrium conditions, the intermediate can be populated to >90%, allowing characterization on a residue-by-residue level using NMR spectroscopy. These data clearly show that the three C-terminal ARs are natively folded in the intermediate state, whereas native crosspeaks for the rest of the molecule are missing. Therefore, the formation of a stable folding unit consisting of three ARs is the necessary rate-limiting step before AR 1 and 2 can assemble to form the native state.
Nature Structural & Molecular Biology, 2005
Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in... more Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in many protein folding reactions.
Journal of Molecular Biology, 2004
ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolo... more ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolobus islandicus. This DNA binding protein is encoded on plasmid pRN1 and possibly controls the copy number of the plasmid. We report the solution NMR structure as well as the crystal structure of ORF56 comprising a ribbon-helix-helix fold. The homodimer consists of an antiparallel intersubunit β-sheet and two R-helices per monomer, which is a common DNA binding fold of plasmid-and phage-encoded gene regulation proteins. NMR titration experiments with ORF56 and double-stranded DNA derived from its promoter binding site revealed that it is largely the β-sheets that interact with the DNA. The β-sheet experiences high local fluctuations, which are conserved among DNA binding ribbon-helix-helix dimers from mesophilic and hyperthermophilic organisms. In contrast, residues strongly protected against H-D exchange are localized in helix 2, forming the hydrophobic intermolecular core of the dimer. A structure-based comparison of the intermolecular binding surface and the change in accessible surface area upon unfolding of various ribbon-helix-helix dimers with the Gibbs free energy changes and m values show a correlation between hydrophobicity of these surface areas and stability. These findings provide possible explanations for the very high thermodynamic stability of ORF56 with retained DNA binding capacity.
Journal of Molecular Biology, 2002
P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase in... more P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.
Journal of Molecular Biology, 2005
The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus ... more The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregationprone proteins.
Journal of Molecular Biology, 2007
The p19 INK4d protein consists of five ankyrin repeats (ANK) and controls the human cell cycle by... more The p19 INK4d protein consists of five ankyrin repeats (ANK) and controls the human cell cycle by inhibiting the cyclin D-dependent kinases (CDK) 4 and 6. We investigated the folding of p19 INK4d by urea-induced unfolding transitions, kinetic analyses of unfolding and refolding, including doublemixing experiments and a special assay for folding intermediates. Folding is a sequential two-step reaction via a hyperfluorescent on-pathway intermediate. This intermediate is present under all conditions, during unfolding, refolding and at equilibrium. The folding mechanism was confirmed by a quantitative global fit of a consistent set of equilibrium and kinetic data revealing the thermodynamics and intrinsic folding rates of the different states. Surprisingly, the N ↔ I transition is much faster compared to the I ↔ U transition. The urea-dependence of the intrinsic folding rates causes population of the intermediate at equilibrium close to the transition midpoint. NMR detected hydrogen/deuterium exchange and the analysis of truncated variants showed that the C-terminal repeats ANK3-5 are already folded in the on-pathway intermediate, whereas the N-terminal repeats 1 and 2 are not folded. We suggest that during refolding, repeats ANK3-ANK5 first form the scaffold for the subsequent assembly of repeats ANK1 and ANK2. The binding function of p19 INK4d resides in the latter repeats. We propose that the graded stability and the facile unfolding of repeats 1 and 2 is a prerequisite for the down-regulation of the inhibitory activity of p19 INK4d during the cell-cycle.
European Biophysics Journal, 2006
Cold and heat denaturation of the double mutant Arg 3 fi Glu/Leu 66 fi Glu of cold shock protein ... more Cold and heat denaturation of the double mutant Arg 3 fi Glu/Leu 66 fi Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D 1 H NMR spectroscopy in the temperature range from À12°C in supercooled water up to +70°C. The fraction of unfolded protein, f u , was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be À11°C and 39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, DC p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.
Biochemistry, 2009
ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolo... more ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolobus islandicus. This DNA binding protein is encoded on plasmid pRN1 and possibly controls the copy number of the plasmid. We report the solution NMR structure as well as the crystal structure of ORF56 comprising a ribbon-helix-helix fold. The homodimer consists of an antiparallel intersubunit beta-sheet and two alpha-helices per monomer, which is a common DNA binding fold of plasmid- and phage-encoded gene regulation proteins. NMR titration experiments with ORF56 and double-stranded DNA derived from its promoter binding site revealed that it is largely the beta-sheets that interact with the DNA. The beta-sheet experiences high local fluctuations, which are conserved among DNA binding ribbon-helix-helix dimers from mesophilic and hyperthermophilic organisms. In contrast, residues strongly protected against H-D exchange are localized in helix 2, forming the hydrophobic intermolecular core of the dimer. A structure-based comparison of the intermolecular binding surface and the change in accessible surface area upon unfolding of various ribbon-helix-helix dimers with the Gibbs free energy changes and m values show a correlation between hydrophobicity of these surface areas and stability. These findings provide possible explanations for the very high thermodynamic stability of ORF56 with retained DNA binding capacity.
Accounts of Chemical Research, 1998
... Anywhere Acc. Chem. Res. All Publications/Website. Select a Journal or Book Series ...
ABSTRACT Supramolecular polyisobutylenes (PIB) bearing mono- and bifunctional chain ends with hyd... more ABSTRACT Supramolecular polyisobutylenes (PIB) bearing mono- and bifunctional chain ends with hydrogen-bonding units were prepared, and their association behavior in the melt state was investigated by dynamic rheology and compared to aggregation in solution, aiming at determining association dynamics in the solid state. A preparation combining living cationic polymerization with either azide/alkyne "click" reactions or nucleophilic substitution reactions enabled a full end group transformation to the final PIB polymers, modified with either thymine or 2,6-diaminotriazine end groups as proven by NMR and MALDI methods with molecular weights of similar to 3500 and similar to 10 000 g/mol. Stoichiometric mixtures of these polymers bearing specifically interacting thymine/triazine moieties were prepared by solution blending and the temperature-dependent dynamics investigated by rheological measurements. At temperatures of 20-60 degrees C all samples display strongly thermoreversible aggregation with sheet-type or partially cross-linked structures, which deaggregate at temperatures of similar to 80 degrees C. More complex aggregates with bridged micellar structure were formed from the respective bifunctional PIB's bearing thymine and 2,6-diaminotriazine moieties. Thus, in addition to specific linear aggregates, the formation of clusters and aggregates of different architecture has to be taken into account to understand and control structure and mechanical properties of supramolecular chains in the melt.
Chemistry and physics of lipids, Jan 29, 2015
Polar lipid pattern determination is often used for the taxonomic classification of halophilic Ar... more Polar lipid pattern determination is often used for the taxonomic classification of halophilic Archaea in addition to a genomic characterization. During the analysis of polar lipid extracts from the recently described haloarchaeon Natrononomonas moolapensis, an unknown glycolipid was detected. Fragmentation patterns observed from preliminary mass spectrometric analysis initially suggested the presence of a sulfo-hexosyl-phosphatidylglycerol. However, by NMR spectroscopy and enzymatic assays the existence of two isomeric molecules with different hexoses (1-(6-sulfo-d-glcp/galf-β1,2-glycero)-phospho-2,3-diphytanylglycerol) could be shown. The structural origin from phosphatidylglycerol distinguishes these glycolipids within Archaea, because all other characterized haloarchaeal glycolipids consist of diphytanylglycerol directly linked to an oligoglycosyl moiety. Now the door is open to investigate the physical and functional consequences of these architectural differences of the head g...
FEBS letters, Jan 19, 2015
d-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-... more d-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-positive bacteria cell walls. The d-alanyl carrier protein DltC from Bacillus subtilis has been solved in apo- and two cofactor-modified holo-forms, whereby the entire phosphopantetheine moiety is defined in one. The atomic resolution of the apo-structure allows delineation of alternative conformations within the hydrophobic core of the 78 residue four helix bundle. In contrast to previous reports for a peptidyl carrier protein from a non-ribosomal peptide synthetase, no obvious structural differences between apo- and holo-DltC forms are observed. Solution NMR spectroscopy confirms these findings and demonstrates in addition that the two forms exhibit similar backbone dynamics on the ps-ns and ms timescales.
Proceedings of the National Academy of Sciences, 2015
wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submi... more wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: The NMR, atomic coordinates, chemical shifts, and restraints have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2M1M), and the Biological Magnetic Resonance Data Bank, www.bmrb.wisc.edu/ (BMRB accession no. 18870).
![Research paper thumbnail of Corrigendum to “High yield production of recombinant native and modified peptides exemplified by ligands for G-protein coupled receptors” [Protein Expr. Purif. 58 (2008) 114–121]](https://attachments.academia-assets.com/46885741/thumbnails/1.jpg)
](Protein Expression and Purification, 2010
The authors discovered an error in the above article. On page 117, in the third line from the top... more The authors discovered an error in the above article. On page 117, in the third line from the top of the first column, the TFA content of solvent A and solvent B was indicated as 0.01%. It should be 0.1%. For the reader's convenience, the corrected sentence appears below.
Biophysical Journal, 2015
Knowledge about the global translational and rotational motion of proteins under crowded conditio... more Knowledge about the global translational and rotational motion of proteins under crowded conditions is highly relevant for understanding the function of proteins in vivo. This holds in particular for human αB-crystallin, which is strongly crowded in vivo and inter alia responsible for preventing cataracts. Quantitative information on translational and rotational diffusion is not readily available, and we here demonstrate an approach that combines pulsed-field-gradient NMR for translational diffusion and proton T1ρ/T2 relaxation-time measurements for rotational diffusion, thus overcoming obstacles encountered in previous studies. The relaxation times measured at variable temperature provide a quantitative measure of the correlation function of protein tumbling, which cannot be approximated by a single exponential, because two components are needed for a minimal and adequate description of the data. We find that at high protein concentrations, rotational diffusion is decoupled from translational diffusion, the latter following the macroscopic viscosity change almost quantitatively, resembling the behavior of spherical colloids. Analysis of data reported in the literature shows that well-packed globular proteins follow a scaling relation between the hydrodynamic radius and the molar mass, Rh ∼ M(1/d), with a fractal dimension of d ∼ 2.5 rather than 3. Despite its oligomeric nature, Rh of αB-crystallin as derived from both NMR methods is found to be fully consistent with this relation.
Structure (London, England : 1993), Jan 6, 2015
The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer t... more The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer toxin insensitive gene KTI11/DPH3 is involved in the diphthamide modification of eukaryotic elongation factor 2 and, together with Kti13, in Elongator-dependent tRNA wobble base modifications, thereby affecting the speed and accuracy of protein biosynthesis through two distinct mechanisms. We have solved the crystal structures of Saccharomyces cerevisiae Kti13 and the Kti11/Kti13 heterodimer at 2.4 and 2.9 Å resolution, respectively, and validated interacting residues through mutational analysis in vitro and in vivo. We show that metal coordination by Kti11 and its heterodimerization with Kti13 are essential for both translational control mechanisms. Our structural and functional analyses identify Kti13 as an additional component of the diphthamide modification pathway and provide insight into the molecular mechanisms that allow the Kti11/Kti13 heterodimer to coregulate two consecutive st...
PLOS ONE, 2015
The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members se... more The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.
Nature Structural Biology, 1995
Proceedings of the National Academy of Sciences, 1997
Protein folding can be described in terms of the development of specific contacts between residue... more Protein folding can be described in terms of the development of specific contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with ␣-lactalbumin reveal the existence of extensive NOEs between aromatic and aliphatic protons in the archetypal molten globule formed by this protein at low pH. Analysis of their time development provides direct evidence for near-native compactness of this state. Through a rapid refolding procedure the NOE intensity can be transferred efficiently into the resolved and assigned spectrum of the native state. This demonstrates the viability of using this approach to map out time-averaged interactions between residues in a partially folded protein.
Proceedings of the National Academy of Sciences, 2008
Repeat proteins are widespread in nature, with many of them functioning as binding molecules in p... more Repeat proteins are widespread in nature, with many of them functioning as binding molecules in protein-protein recognition. Their simple structural architecture is used in biotechnology for generating proteins with high affinities to target proteins. Recent folding studies of ankyrin repeat (AR) proteins revealed a new mechanism of protein folding. The formation of an intermediate state is rate limiting in the folding reaction, suggesting a scaffold function of this transient state for intrinsically less stable ARs. To investigate a possible common mechanism of AR folding, we studied the structure and folding of a new thermophilic AR protein (tANK) identified in the archaeon Thermoplasma volcanium. The x-ray structure of the evolutionary much older tANK revealed high homology to the human CDK inhibitor p19 INK4d , whose sequence was used for homology search. As for p19 INK4d , equilibrium and kinetic folding analyses classify tANK to the family of sequential three-state folding proteins, with an unusual fast equilibrium between native and intermediate state. Under equilibrium conditions, the intermediate can be populated to >90%, allowing characterization on a residue-by-residue level using NMR spectroscopy. These data clearly show that the three C-terminal ARs are natively folded in the intermediate state, whereas native crosspeaks for the rest of the molecule are missing. Therefore, the formation of a stable folding unit consisting of three ARs is the necessary rate-limiting step before AR 1 and 2 can assemble to form the native state.
Nature Structural & Molecular Biology, 2005
Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in... more Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in many protein folding reactions.
Journal of Molecular Biology, 2004
ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolo... more ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolobus islandicus. This DNA binding protein is encoded on plasmid pRN1 and possibly controls the copy number of the plasmid. We report the solution NMR structure as well as the crystal structure of ORF56 comprising a ribbon-helix-helix fold. The homodimer consists of an antiparallel intersubunit β-sheet and two R-helices per monomer, which is a common DNA binding fold of plasmid-and phage-encoded gene regulation proteins. NMR titration experiments with ORF56 and double-stranded DNA derived from its promoter binding site revealed that it is largely the β-sheets that interact with the DNA. The β-sheet experiences high local fluctuations, which are conserved among DNA binding ribbon-helix-helix dimers from mesophilic and hyperthermophilic organisms. In contrast, residues strongly protected against H-D exchange are localized in helix 2, forming the hydrophobic intermolecular core of the dimer. A structure-based comparison of the intermolecular binding surface and the change in accessible surface area upon unfolding of various ribbon-helix-helix dimers with the Gibbs free energy changes and m values show a correlation between hydrophobicity of these surface areas and stability. These findings provide possible explanations for the very high thermodynamic stability of ORF56 with retained DNA binding capacity.
Journal of Molecular Biology, 2002
P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase in... more P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.
Journal of Molecular Biology, 2005
The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus ... more The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregationprone proteins.
Journal of Molecular Biology, 2007
The p19 INK4d protein consists of five ankyrin repeats (ANK) and controls the human cell cycle by... more The p19 INK4d protein consists of five ankyrin repeats (ANK) and controls the human cell cycle by inhibiting the cyclin D-dependent kinases (CDK) 4 and 6. We investigated the folding of p19 INK4d by urea-induced unfolding transitions, kinetic analyses of unfolding and refolding, including doublemixing experiments and a special assay for folding intermediates. Folding is a sequential two-step reaction via a hyperfluorescent on-pathway intermediate. This intermediate is present under all conditions, during unfolding, refolding and at equilibrium. The folding mechanism was confirmed by a quantitative global fit of a consistent set of equilibrium and kinetic data revealing the thermodynamics and intrinsic folding rates of the different states. Surprisingly, the N ↔ I transition is much faster compared to the I ↔ U transition. The urea-dependence of the intrinsic folding rates causes population of the intermediate at equilibrium close to the transition midpoint. NMR detected hydrogen/deuterium exchange and the analysis of truncated variants showed that the C-terminal repeats ANK3-5 are already folded in the on-pathway intermediate, whereas the N-terminal repeats 1 and 2 are not folded. We suggest that during refolding, repeats ANK3-ANK5 first form the scaffold for the subsequent assembly of repeats ANK1 and ANK2. The binding function of p19 INK4d resides in the latter repeats. We propose that the graded stability and the facile unfolding of repeats 1 and 2 is a prerequisite for the down-regulation of the inhibitory activity of p19 INK4d during the cell-cycle.
European Biophysics Journal, 2006
Cold and heat denaturation of the double mutant Arg 3 fi Glu/Leu 66 fi Glu of cold shock protein ... more Cold and heat denaturation of the double mutant Arg 3 fi Glu/Leu 66 fi Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D 1 H NMR spectroscopy in the temperature range from À12°C in supercooled water up to +70°C. The fraction of unfolded protein, f u , was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be À11°C and 39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, DC p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.
Biochemistry, 2009
ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolo... more ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolobus islandicus. This DNA binding protein is encoded on plasmid pRN1 and possibly controls the copy number of the plasmid. We report the solution NMR structure as well as the crystal structure of ORF56 comprising a ribbon-helix-helix fold. The homodimer consists of an antiparallel intersubunit beta-sheet and two alpha-helices per monomer, which is a common DNA binding fold of plasmid- and phage-encoded gene regulation proteins. NMR titration experiments with ORF56 and double-stranded DNA derived from its promoter binding site revealed that it is largely the beta-sheets that interact with the DNA. The beta-sheet experiences high local fluctuations, which are conserved among DNA binding ribbon-helix-helix dimers from mesophilic and hyperthermophilic organisms. In contrast, residues strongly protected against H-D exchange are localized in helix 2, forming the hydrophobic intermolecular core of the dimer. A structure-based comparison of the intermolecular binding surface and the change in accessible surface area upon unfolding of various ribbon-helix-helix dimers with the Gibbs free energy changes and m values show a correlation between hydrophobicity of these surface areas and stability. These findings provide possible explanations for the very high thermodynamic stability of ORF56 with retained DNA binding capacity.
Accounts of Chemical Research, 1998
... Anywhere Acc. Chem. Res. All Publications/Website. Select a Journal or Book Series ...
ABSTRACT Supramolecular polyisobutylenes (PIB) bearing mono- and bifunctional chain ends with hyd... more ABSTRACT Supramolecular polyisobutylenes (PIB) bearing mono- and bifunctional chain ends with hydrogen-bonding units were prepared, and their association behavior in the melt state was investigated by dynamic rheology and compared to aggregation in solution, aiming at determining association dynamics in the solid state. A preparation combining living cationic polymerization with either azide/alkyne "click" reactions or nucleophilic substitution reactions enabled a full end group transformation to the final PIB polymers, modified with either thymine or 2,6-diaminotriazine end groups as proven by NMR and MALDI methods with molecular weights of similar to 3500 and similar to 10 000 g/mol. Stoichiometric mixtures of these polymers bearing specifically interacting thymine/triazine moieties were prepared by solution blending and the temperature-dependent dynamics investigated by rheological measurements. At temperatures of 20-60 degrees C all samples display strongly thermoreversible aggregation with sheet-type or partially cross-linked structures, which deaggregate at temperatures of similar to 80 degrees C. More complex aggregates with bridged micellar structure were formed from the respective bifunctional PIB's bearing thymine and 2,6-diaminotriazine moieties. Thus, in addition to specific linear aggregates, the formation of clusters and aggregates of different architecture has to be taken into account to understand and control structure and mechanical properties of supramolecular chains in the melt.
Chemistry and physics of lipids, Jan 29, 2015
Polar lipid pattern determination is often used for the taxonomic classification of halophilic Ar... more Polar lipid pattern determination is often used for the taxonomic classification of halophilic Archaea in addition to a genomic characterization. During the analysis of polar lipid extracts from the recently described haloarchaeon Natrononomonas moolapensis, an unknown glycolipid was detected. Fragmentation patterns observed from preliminary mass spectrometric analysis initially suggested the presence of a sulfo-hexosyl-phosphatidylglycerol. However, by NMR spectroscopy and enzymatic assays the existence of two isomeric molecules with different hexoses (1-(6-sulfo-d-glcp/galf-β1,2-glycero)-phospho-2,3-diphytanylglycerol) could be shown. The structural origin from phosphatidylglycerol distinguishes these glycolipids within Archaea, because all other characterized haloarchaeal glycolipids consist of diphytanylglycerol directly linked to an oligoglycosyl moiety. Now the door is open to investigate the physical and functional consequences of these architectural differences of the head g...
FEBS letters, Jan 19, 2015
d-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-... more d-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-positive bacteria cell walls. The d-alanyl carrier protein DltC from Bacillus subtilis has been solved in apo- and two cofactor-modified holo-forms, whereby the entire phosphopantetheine moiety is defined in one. The atomic resolution of the apo-structure allows delineation of alternative conformations within the hydrophobic core of the 78 residue four helix bundle. In contrast to previous reports for a peptidyl carrier protein from a non-ribosomal peptide synthetase, no obvious structural differences between apo- and holo-DltC forms are observed. Solution NMR spectroscopy confirms these findings and demonstrates in addition that the two forms exhibit similar backbone dynamics on the ps-ns and ms timescales.
Proceedings of the National Academy of Sciences, 2015
wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submi... more wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: The NMR, atomic coordinates, chemical shifts, and restraints have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2M1M), and the Biological Magnetic Resonance Data Bank, www.bmrb.wisc.edu/ (BMRB accession no. 18870).
Protein Expression and Purification, 2010
The authors discovered an error in the above article. On page 117, in the third line from the top... more The authors discovered an error in the above article. On page 117, in the third line from the top of the first column, the TFA content of solvent A and solvent B was indicated as 0.01%. It should be 0.1%. For the reader's convenience, the corrected sentence appears below.
Biophysical Journal, 2015
Knowledge about the global translational and rotational motion of proteins under crowded conditio... more Knowledge about the global translational and rotational motion of proteins under crowded conditions is highly relevant for understanding the function of proteins in vivo. This holds in particular for human αB-crystallin, which is strongly crowded in vivo and inter alia responsible for preventing cataracts. Quantitative information on translational and rotational diffusion is not readily available, and we here demonstrate an approach that combines pulsed-field-gradient NMR for translational diffusion and proton T1ρ/T2 relaxation-time measurements for rotational diffusion, thus overcoming obstacles encountered in previous studies. The relaxation times measured at variable temperature provide a quantitative measure of the correlation function of protein tumbling, which cannot be approximated by a single exponential, because two components are needed for a minimal and adequate description of the data. We find that at high protein concentrations, rotational diffusion is decoupled from translational diffusion, the latter following the macroscopic viscosity change almost quantitatively, resembling the behavior of spherical colloids. Analysis of data reported in the literature shows that well-packed globular proteins follow a scaling relation between the hydrodynamic radius and the molar mass, Rh ∼ M(1/d), with a fractal dimension of d ∼ 2.5 rather than 3. Despite its oligomeric nature, Rh of αB-crystallin as derived from both NMR methods is found to be fully consistent with this relation.
Structure (London, England : 1993), Jan 6, 2015
The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer t... more The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer toxin insensitive gene KTI11/DPH3 is involved in the diphthamide modification of eukaryotic elongation factor 2 and, together with Kti13, in Elongator-dependent tRNA wobble base modifications, thereby affecting the speed and accuracy of protein biosynthesis through two distinct mechanisms. We have solved the crystal structures of Saccharomyces cerevisiae Kti13 and the Kti11/Kti13 heterodimer at 2.4 and 2.9 Å resolution, respectively, and validated interacting residues through mutational analysis in vitro and in vivo. We show that metal coordination by Kti11 and its heterodimerization with Kti13 are essential for both translational control mechanisms. Our structural and functional analyses identify Kti13 as an additional component of the diphthamide modification pathway and provide insight into the molecular mechanisms that allow the Kti11/Kti13 heterodimer to coregulate two consecutive st...