Publications by authors named "Jochen Balbach"

96 Publications

Inactivation of parathyroid hormone: perspectives of drug discovery to combating hyperparathyroidism.

Curr Mol Pharmacol 2021 Jan 25. Epub 2021 Jan 25.

Institute of Physics, Biophysics, Martin-Luther-University Halle- Wittenberg. Germany.

Hormonal coordination is tightly regulated within the human body and thus regulates human physiology. The parathyroid hormone (PTH), a member of the endocrine system, regulates the calcium and phosphate level within the human body. Under non-physiological conditions, PTH levels get upregulated (hyperparathyroidism) or downregulated (hypoparathyroidism) due to external or internal factors. In the case of hyperparathyroidism, elevated PTH stimulates cellular receptors present in the bones, kidneys, and intestines to increase the blood calcium level, leading to calcium deposition. This eventually causes various symptoms including kidney stones. Currently, there is no known medication that directly targets PTH in order to suppress its function. Therefore, it is of great interest to find novel small molecules or any other means that can modulate PTH function. The molecular signaling of PTH starts by binding of its N-terminus to the G-protein coupled PTH1/2 receptor. Therefore, any intervention that affects the N-terminus of PTH could be a lead candidate for treating hyperparathyroidism. As a proof-of-concept, there are various possibilities to inhibit molecular PTH function by (i) a small molecule, (ii) N-terminal PTH phosphorylation, (iii) fibril formation and (iv) residue-specific mutations. These modifications put PTH into an inactive state, which will be discussed in detail in this review article. We anticipate that exploring small molecules or other means that affect the N-terminus of PTH could be lead candidates in combating hyperparathyroidism.
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http://dx.doi.org/10.2174/1874467214666210126112839DOI Listing
January 2021

Lipid-Dependent Interaction of Human N-BAR Domain Proteins with Sarcolemma Mono- and Bilayers.

Langmuir 2020 08 23;36(30):8695-8704. Epub 2020 Jul 23.

Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Straße 3a, 06120 Halle (Saale), Germany.

The N-BAR domain of the human Bin1 protein is indispensable for T-tubule biogenesis in skeletal muscles. It binds to lipid mono- and bilayers that mimic the sarcolemma membrane composition, and it transforms vesicles into uniform tubules by generating a decorating protein scaffold. We found that Δ(1-33)BAR, lacking the N-terminal amphipathic helix (H0), and H0 alone bind to sarcolemma monolayers, although both proteins are not able to tubulate sarcolemma vesicles. By variation of the lipid composition, we elucidated the role of PI(4,5)P, cholesterol, and an asymmetric sarcolemma composition for Bin1-N-BAR binding and sarcolemma tubulation. Our results indicate that Bin1-N-BAR binding is low in the absence of PI(4,5)P and it is affected by additional changes in the negative headgroup charge and lipid acyl chain composition. However, it is not dependent on the cholesterol content. The results from Langmuir monolayer experiments are complementary to lipid bilayer studies using electron microscopy that provides information on membrane curvature generation.
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http://dx.doi.org/10.1021/acs.langmuir.0c00649DOI Listing
August 2020

Insights into the secondary structures of lactam N-substituted stapled peptides.

Org Biomol Chem 2020 May;18(20):3838-3842

Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120, Halle/Saale, Germany. and Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, 10400, Havana, Cuba.

Stapled peptides derived from the Ugi macrocyclization comprise a special class of cyclopeptides with an N-substituted lactam bridge cross-linking two amino acid side chains. Herein we report a comprehensive analysis of the structural factors influencing the secondary structure of these cyclic peptides in solution. Novel insights into the s-cis/s-trans isomerism and the effect of N-functionalization on the conformation are revealed.
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http://dx.doi.org/10.1039/d0ob00767fDOI Listing
May 2020

Synthesis and Aggregation of Polymer-Amyloid β Conjugates.

Macromol Rapid Commun 2020 Jan 21;41(1):e1900378. Epub 2019 Oct 21.

Faculty of Natural Science II, Institute of Chemistry, Martin-Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, D-06120, Halle (Saale), Germany.

Modulating the assembly of medically relevant peptides and proteins via macromolecular engineering is an important step in modifying their overall pathological effects. The synthesis of polymer-peptide conjugates composed of the amyloidogenic Alzheimer peptide, Aβ , and poly(oligo(ethylene glycol) acrylates) (m = 2,3) with different molecular weights (M = 1400-6600 g mol ) is presented here. The challenging conjugation of a synthetic polymer to an in situ aggregating protein is established via two different coupling strategies, only successful for polymers with molecular weights not exceeding 6600 g mol , relying on resin-based synthesis or solution-based coupling chemistries. The conjugates are characterized by high-performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The aggregation of these polymer-Aβ conjugates, as monitored via thioflavine-T (ThT)-fluorescence spectroscopy, is accelerated mainly upon attaching the polymers. However, the appearance of the observed fibrils is different from those composed of native Aβ specifically with respect to length and morphology of the obtained aggregates. Instead of long, unbranched fibrils characteristic for Aβ , bundles of short aggregates are observed for the conjugates. Finally, the ThT kinetics and morphologies of Aβ fibrils formed in the presence of the conjugates give some mechanistic insights.
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http://dx.doi.org/10.1002/marc.201900378DOI Listing
January 2020

Modulation of amyloid β peptide aggregation by hydrophilic polymers.

Phys Chem Chem Phys 2019 Oct 17;21(37):20999-21006. Epub 2019 Sep 17.

Martin-Luther University Halle-Wittenberg, Faculty of Natural Science II, Institute of Chemistry, Von-Danckelmann-Platz 4, D-06120 Halle (Saale), Germany.

A substantial number of diseases leading to loss of neurologic functions such as Morbus Alzheimer, Morbus Parkinson, or Chorea Huntington are related to the fibrillation of particular amyloidogenic peptides. In vitro amyloid fibrillation strongly depends on admixture with other proteins and peptides, lipids, nanoparticles, surfactants and polymers. We investigated amyloid-beta 1-40 peptide (Aβ) fibrillation in mixture with thermoresponsive poly(oligo(ethylene glycol)acrylates), in which the polymer's hydrophobicity is tuned by variation of the number of ethylene glycol-units in the side chain (m = 1-9), the end groups (B = butoxy; C = carboxy; D = dodecyl; P = pyridyldisulfide) and the degree of polymerization (n) of the polymers. The polymers were prepared via RAFT-polymerization, obtaining a broad range of molecular masses (M = 700 to 14 600 g mol kDa, polydispersity indices PDI = 1.10 to 1.25) and tunable cloud point temperatures (T), ranging from 42.4 °C to 80 °C, respectively. Proper combination of hydrophobic end groups with hydrophilic side chains of the polymer allowed to alter the hydrophilicity/hydrophobicity of these polymers, which is shown to enhance Aβ aggregation significantly in case of the endgroup D (with n = 16, 23, 56). We observed that the less hydrophilic polymers (m = 1-2) were able to both decrease and elongate the lag (tlag) and characteristic times (tchar) of Aβ fibril formation in dependence of their end groups, molecular mass and hydrophilicity. On the other hand, highly hydrophilic polymers (m = 3, 5, 9) either decreased, or only marginally influenced the lag and characteristic times of Aβ fibrillation, in all cases forming β-sheet rich fibrils as observed by TEM and CD-spectroscopy. Our results support that balanced hydrophobic and hydrophilic interactions of a polymer with Aβ is important for inhibiting amyloid-formation pathways.
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http://dx.doi.org/10.1039/c9cp02683eDOI Listing
October 2019

Corrigendum to quantitative NMR study of heat-induced aggregation of eye-lens crystallin proteins under crowding conditions. BBAPAP Volume 1866/10 2018 1055-1061].

Biochim Biophys Acta Proteins Proteom 2019 Apr 14;1867(4):453-454. Epub 2019 Feb 14.

Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle (Saale), Germany. Electronic address:

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http://dx.doi.org/10.1016/j.bbapap.2019.02.001DOI Listing
April 2019

In-Cell NMR: Analysis of Protein-Small Molecule Interactions, Metabolic Processes, and Protein Phosphorylation.

Int J Mol Sci 2019 01 17;20(2). Epub 2019 Jan 17.

Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany.

Nuclear magnetic resonance (NMR) spectroscopy enables the non-invasive observation of biochemical processes, in living cells, at comparably high spectral and temporal resolution. Preferably, means of increasing the detection limit of this powerful analytical method need to be applied when observing cellular processes under physiological conditions, due to the low sensitivity inherent to the technique. In this review, a brief introduction to in-cell NMR, protein-small molecule interactions, posttranslational phosphorylation, and hyperpolarization NMR methods, used for the study of metabolites in cellulo, are presented. Recent examples of method development in all three fields are conceptually highlighted, and an outlook into future perspectives of this emerging area of NMR research is given.
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http://dx.doi.org/10.3390/ijms20020378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359726PMC
January 2019

How Fluorescent Tags Modify Oligomer Size Distributions of the Alzheimer Peptide.

Biophys J 2019 01 19;116(2):227-238. Epub 2018 Dec 19.

Department of Biotechnology and Biochemistry, Martin-Luther-University Halle-Wittenberg, Halle, Germany. Electronic address:

Within the complex aggregation process of amyloidogenic peptides into fibrils, early stages of aggregation play a central role and reveal fundamental properties of the underlying mechanism of aggregation. In particular, low-molecular-weight aggregates of the Alzheimer amyloid-β peptide (Aβ) have attracted increasing interest because of their role in cytotoxicity and neuronal apoptosis, typical of aggregation-related diseases. One of the main techniques used to characterize oligomeric stages is fluorescence spectroscopy. To this end, Aβ peptide chains are functionalized with fluorescent tags, often covalently bound to the disordered N-terminus region of the peptide, with the assumption that functionalization and presence of the fluorophore will not modify the process of self-assembly nor the final fibrillar structure. In this investigation, we systematically study the effects of four of the most commonly used fluorophores on the aggregation of Aβ (1-40). Time-resolved and single-molecule fluorescence spectroscopy have been chosen to monitor the oligomer populations at different fibrillation times, and transmission electron microscopy, atomic force microscopy and x-ray diffraction to investigate the structure of mature fibrils. Although the structures of the fibrils were only slightly affected by the fluorescent tags, the sizes of the detected oligomeric species varied significantly depending on the chosen fluorophore. In particular, we relate the presence of high-molecular-weight oligomers of Aβ (1-40) (as found for the fluorophores HiLyte 647 and Atto 655) to net-attractive, hydrophobic fluorophore-peptide interactions, which are weak in the case of HiLyte 488 and Atto 488. The latter leads for Aβ (1-40) to low-molecular-weight oligomers only, which is in contrast to Aβ (1-42). The disease-relevant peptide Aβ (1-42) displays high-molecular-weight oligomers even in the absence of significant attractive fluorophore-peptide interactions. Hence, our findings reveal the potentially high impact of the properties of fluorophores on transient aggregates, which needs to be included in the interpretation of experimental data of oligomers of fluorescently labeled peptides.
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http://dx.doi.org/10.1016/j.bpj.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350010PMC
January 2019

Hyperbolic Pressure-Temperature Phase Diagram of the Zinc-Finger Protein apoKti11 Detected by NMR Spectroscopy.

J Phys Chem B 2019 01 16;123(4):792-801. Epub 2019 Jan 16.

Institute of Physics, Biophysics , Martin-Luther University Halle-Wittenberg , Betty-Heimann Street 7 , 06120 Halle , Germany.

For a comprehensive understanding of the thermodynamic state functions describing the stability of a protein, the influence of the intensive properties of temperature and pressure has to be known. With the zinc-finger-containing Kti11, we found a suitable protein for this purpose because folding and unfolding transitions occur at an experimentally accessible temperature (280-330 °K) and pressure (0.1-240 MPa) range. We solved the crystal structure of the apo form of Kti11 to reveal two disulfide bonds at the metal-binding site, which seals off a cavity in the β-barrel part of the protein. From a generally applicable proton NMR approach, we could determine the populations of folded and unfolded chains under all conditions, leading to a hyperbolic pressure-temperature phase diagram rarely observed for proteins. A global fit of a two-state model to all derived populations disclosed reliable values for the change in Gibbs free energy, volume, entropy, heat capacity, compressibility, and thermal expansion upon unfolding. The unfolded state of apoKti11 has a lower compressibility compared to the native state and a smaller volume at ambient pressure. Therefore, a pressure increase up to 200 MPa reduces the population of the native state, and above this value, the native population increases again. Pressure-induced chemical-shift changes in two-dimensional H-N NMR spectra could be employed for a molecular interpretation of the thermodynamic properties of apoKti11.
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http://dx.doi.org/10.1021/acs.jpcb.8b11019DOI Listing
January 2019

A Multicomponent Stapling Approach to Exocyclic Functionalized Helical Peptides: Adding Lipids, Sugars, PEGs, Labels, and Handles to the Lactam Bridge.

Bioconjug Chem 2019 01 7;30(1):253-259. Epub 2019 Jan 7.

Department of Bioorganic Chemistry , Leibniz Institute of Plant Biochemistry , Weinberg 3 , D-06120 , Halle (Saale) , Germany.

Peptide stapling is traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization chemistries. In an endeavor to add a diversity-generating tool to this repertoire, we introduce a multicomponent stapling approach enabling the simultaneous stabilization of helical secondary structures and the exocyclic N-functionalization of the side chain-tethering lactam bridge. This is accomplished by means of a novel solid-phase methodology comprising, for the first time, the on-resin Ugi reaction-based macrocyclization of peptide side chains bearing amino and carboxylic acid groups. The exocyclic diversity elements arise from the isocyanide component used in the Ugi multicomponent stapling protocol, which allows for the incorporation of relevant fragments such as lipids, sugars, polyethylene glycol, fluorescent labels, and reactive handles. We prove the utility of such exocyclic reactive groups in the bioconjugation of a maleimide-armed lactam-bridged peptide to a carrier protein. The on-resin multicomponent stapling proved efficient for the installation of not only one, but also two consecutive lactam bridges having either identical or dissimilar N-functionalities. The easy access to helical peptides with a diverse set of exocyclic functionalities shows prospect for applications in peptide drug discovery and chemical biology.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00906DOI Listing
January 2019

Probing Polymer Chain Conformation and Fibril Formation of Peptide Conjugates.

Chemphyschem 2019 01 13;20(2):236-240. Epub 2018 Nov 13.

Institute of Physics, Martin Luther University Halle-Wittenberg, Betty-Heimann-Str. 7, 06120, Halle (Saale), Germany.

Covalent conjugates between a synthetic polymer and a peptide hormone were used to probe the molecular extension of these macromolecules and how the polymer modifies the fibril formation of the hormone. NMR spectroscopy of N labeled parathyroid hormone (PTH) was employed to visualize the conformation of the conjugated synthetic polymer, triggered by small temperature changes via its lower critical solution temperature. A shroud-like polymer conformation dominated the molecular architecture of the conjugated chimeras. PTH readily forms amyloid fibrils, which is probably the physiological storage form of the hormone. The polyacrylate based polymers stimulated the nucleation processes of the peptide.
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http://dx.doi.org/10.1002/cphc.201800867DOI Listing
January 2019

Equilibrium and Kinetic Unfolding of GB1: Stabilization of the Native State by Pressure.

J Phys Chem B 2018 09 14;122(38):8846-8852. Epub 2018 Sep 14.

Institute of Physics, Biophysics , Martin-Luther-University Halle-Wittenberg , D-06120 Halle (Saale) , Germany.

NMR spectroscopy allows an all-atom view on pressure-induced protein folding, separate detection of different folding states, determination of their population, and the measurement of the folding kinetics at equilibrium. Here, we studied the folding of protein GB1 at pH 2 in a temperature and pressure dependent way. We find that the midpoints of temperature-induced unfolding increase with higher pressure. NMR relaxation dispersion experiments disclosed that the unfolding kinetics slow down at elevated pressure while the folding kinetics stay virtually the same. Therefore, pressure is stabilizing the native state of GB1. These findings extend the knowledge of the influence of pressure on protein folding kinetics, where so far typically a destabilization by increased activation volumes of folding was observed. Our findings thus point toward an exceptional section in the pressure-temperature phase diagram of protein unfolding. The stabilization of the native state could potentially be caused by a shift of p K values of glutamates and aspartates in favor of the negatively charged state as judged from pH sensitive chemical shifts.
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http://dx.doi.org/10.1021/acs.jpcb.8b06888DOI Listing
September 2018

Quantitative NMR study of heat-induced aggregation of eye-lens crystallin proteins under crowding conditions.

Biochim Biophys Acta Proteins Proteom 2018 Jul 30. Epub 2018 Jul 30.

Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle (Saale), Germany. Electronic address:

The eye lens contains a highly concentrated, polydisperse mixture of crystallins, and a loss in transparency during cataract formation is attributed to the aggregation of these proteins. Most biochemical and biophysical studies of crystallins have been performed in diluted samples because of various physical limitations of the respective method at physiological concentrations of up to 200-400 mg/mL. We introduce a straightforward proton NMR transverse relaxometry method to quantify simultaneously proteins in the dissolved and aggregated states at these elevated concentrations, because these states significantly differ in their transverse relaxation properties. The key feature of this method is a direct observation of the protein signal in a wide range of relaxation delays, from few microseconds up to few hundred milliseconds. We applied this method to follow heat-induced aggregation of bovine α- and γB-crystallin between 60 and 200 mg/mL. We find that at 60 °C, a temperature where both crystallins still comprise a native tertiary structure, γB-crystallin aggregated at these high protein concentrations with a time constant of about 30-40 h. α-crystallin remained soluble at 60 mg/mL but formed a transparent gel at 200 mg/mL. This quantitative NMR method can be applied to investigations of other proteins and their mixtures under various aggregation conditions.
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http://dx.doi.org/10.1016/j.bbapap.2018.07.007DOI Listing
July 2018

A Cu complex induces the aggregation of human papillomavirus oncoprotein E6 and stabilizes p53.

FEBS J 2018 08 6;285(16):3013-3025. Epub 2018 Jul 6.

Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, Germany.

Papillomavirus oncoprotein E6 is a critical factor in the modulation of cervical cancer in humans. At the molecular level, formation of the E6-E6AP-p53 ternary complex, which directs p53's degradation, is the key instigator of cancer transforming properties. Herein, a Cu anthracenyl-terpyridine complex is described which specifically induces the aggregation of E6 in vitro and in cultured cells. For a hijacking mechanism, both E6 and E6AP are required for p53 ubiquitination and degradation. The Cu complex interacts with E6 at the E6AP and p53 binding sites. We show that E6 function is suppressed by aggregation, rendering it incapable of hijacking p53 and thus increasing its cellular level. Therapeutic treatments of cervical cancer are currently unavailable to infected individuals. We anticipate that this Cu complex might open up a new therapeutic avenue for the design and development of new chemical entities for the diagnosis and treatment of HPV-induced cancers.
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http://dx.doi.org/10.1111/febs.14591DOI Listing
August 2018

Phosphorylation-induced unfolding regulates p19 during the human cell cycle.

Proc Natl Acad Sci U S A 2018 03 12;115(13):3344-3349. Epub 2018 Mar 12.

Biophysics Group, Institute of Physics, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany;

Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs). The ankyrin-repeat protein p19 functions as a key regulator of G1/S transition; however, its molecular mode of action is unknown. Here, we combine cell and structural biology methods to unravel the mechanism by which p19 controls cell cycle progression. We delineate how the stepwise phosphorylation of p19 Ser66 and Ser76 by cell cycle-independent (p38) and -dependent protein kinases (CDK1), respectively, leads to local unfolding of the three N-terminal ankyrin repeats of p19 This dissociates the CDK6-p19 inhibitory complex and, thereby, activates CDK6. CDK6 triggers entry into S-phase, whereas p19 is ubiquitinated and degraded. Our findings reveal how signaling-dependent p19 unfolding contributes to the irreversibility of G1/S transition.
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http://dx.doi.org/10.1073/pnas.1719774115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879693PMC
March 2018

Structure-Based Insights into the Dynamics and Function of Two-Domain SlpA from Escherichia coli.

Biochemistry 2017 Dec 1;56(50):6533-6543. Epub 2017 Dec 1.

Institut für Physik, Biophysik, Martin-Luther-Universität Halle-Wittenberg , D-06099 Halle (Saale), Germany.

SlpA (SlyD-like protein A) comprises two domains, a FK506 binding domain (FKBP fold) of moderate prolyl cis/trans-isomerase activity and an inserted in flap (IF) domain that hosts its chaperone activity. Here we present the nuclear magnetic resonance (NMR) solution structure of apo Escherichia coli SlpA determined by NMR that mirrors the structural properties seen for various SlyD homologues. Crucial structural differences in side-chain orientation arise for F37, which points directly into the hydrophobic core of the active site. It forms a prominent aromatic stacking with F15, one of the key residues for PPIase activity, thus giving a possible explanation for the inherently low PPIase activity of SlpA. The IF domain reveals the highest stability within the FKBP-IF protein family, most likely arising from an aromatic cluster formed by four phenylalanine residues. Both the thermodynamic stability and the PPIase and chaperone activity let us speculate that SlpA is a backup system for homologous bacterial systems under unfavorable conditions.
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http://dx.doi.org/10.1021/acs.biochem.7b00786DOI Listing
December 2017

Targeting the molecular chaperone SlyD to inhibit bacterial growth with a small molecule.

Sci Rep 2017 02 8;7:42141. Epub 2017 Feb 8.

Institute of Physics, Biophysics, Martin Luther University, Halle, Wittenberg, Germany.

Molecular chaperones are essential molecules for cell growth, whereby they maintain protein homeostasis. Because of their central cellular function, bacterial chaperones might be potential candidates for drug targets. Antimicrobial resistance is currently one of the greatest threats to human health, with gram-negative bacteria being of major concern. We found that a Cu complex readily crosses the bacterial cell wall and inhibits SlyD, which is a molecular chaperone, cis/trans peptidyl prolyl isomerise (PPIase) and involved in various other metabolic pathways. The Cu complex binds to the active sites of SlyD, which suppresses its PPIase and chaperone activities. Significant cell growth retardation could be observed for pathogenic bacteria (e.g., Staphylococcus aureus and Pseudomonas aeruginosa). We anticipate that rational development of drugs targeting molecular chaperones might help in future control of pathogenic bacterial growth, in an era of rapidly increasing antibiotic resistance.
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http://dx.doi.org/10.1038/srep42141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296862PMC
February 2017

Coupling and Decoupling of Rotational and Translational Diffusion of Proteins under Crowding Conditions.

J Am Chem Soc 2016 08 3;138(32):10365-72. Epub 2016 Aug 3.

Institut für Physik, Martin-Luther-Universität Halle-Wittenberg , 06099 Halle (Saale), Germany.

Molecular motion of biopolymers in vivo is known to be strongly influenced by the high concentration of organic matter inside cells, usually referred to as crowding conditions. To elucidate the effect of intermolecular interactions on Brownian motion of proteins, we performed (1)H pulsed-field gradient NMR and fluorescence correlation spectroscopy (FCS) experiments combined with small-angle X-ray scattering (SAXS) and viscosity measurements for three proteins, αB-crystalline (αBc), bovine serum albumin, and hen egg-white lysozyme (HEWL) in aqueous solution. Our results demonstrate that long-time translational diffusion quantitatively follows the expected increase of macro-viscosity upon increasing the protein concentration in all cases, while rotational diffusion as assessed by polarized FCS and previous multi-frequency (1)H NMR relaxometry experiments reveals protein-specific behavior spanning the full range between the limiting cases of full decoupling from (αBc) and full coupling to (HEWL) the macro-viscosity. SAXS was used to study the interactions between the proteins in solution, whereby it is shown that the three cases cover the range between a weakly interacting hard-sphere system (αBc) and screened Coulomb repulsion combined with short-range attraction (HEWL). Our results, as well as insights from the recent literature, suggest that the unusual rotational-translational coupling may be due to anisotropic interactions originating from hydrodynamic shape effects combined with high charge and possibly a patchy charge distribution.
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http://dx.doi.org/10.1021/jacs.6b06615DOI Listing
August 2016

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid β (1-40).

Chemphyschem 2016 Sep 27;17(17):2744-53. Epub 2016 Jun 27.

Institute for Medical Physics and Biophysics, Leipzig University, Härtelstrasse 16-18, 04107, Leipzig, Germany.

A small library of rationally designed amyloid β [Aβ(1-40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aβ(1-40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid-state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aβ(1-40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aβ fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross-β structure.
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http://dx.doi.org/10.1002/cphc.201600413DOI Listing
September 2016

Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane.

J Struct Biol 2016 06 22;194(3):375-82. Epub 2016 Mar 22.

Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine, ZIK HALOmem, Martin Luther University Halle-Wittenberg, 06120 Halle, Germany. Electronic address:

The 30kDa N-BAR domain of the human Bin1 protein is essential for the generation of skeletal muscle T-tubules. By electron cryo-microscopy and electron cryo-tomography with a direct electron detector, we found that Bin1-N-BAR domains assemble into scaffolds of low long-range order that form flexible membrane tubules. The diameter of the tubules closely matches the curved shape of the N-BAR domain, which depends on the composition of the target membrane. These insights are fundamental to our understanding of T-tubule formation and function in human skeletal muscle.
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http://dx.doi.org/10.1016/j.jsb.2016.03.017DOI Listing
June 2016

Inhibition of Aβ(1-40) fibril formation by cyclophilins.

Biochem J 2016 05 18;473(10):1355-68. Epub 2016 Mar 18.

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle (Saale), Germany Department of Enzymology, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Weinbergweg 22, D-06120 Halle (Saale), Germany

Cyclophilins interact directly with the Alzheimer's disease peptide Aβ (amyloid β-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aβ binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aβ(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aβ(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aβ(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aβ(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aβ fibril formation by cyclophilins.
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http://dx.doi.org/10.1042/BCJ20160098DOI Listing
May 2016

Molecular architecture of Aβ fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aβ plaque formation.

Amyloid 2016 Jun 14;23(2):76-85. Epub 2016 Mar 14.

b Institute for Physics, Biophysics, Martin-Luther-University Halle-Wittenberg , Halle (Saale) , Germany .

Objectives: The detailed structure of brain-derived Aβ amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aβ(1-40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aβ amyloid plaque formation.

Methods: We have used hydrogen-deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aβ fibrils.

Results And Conclusions: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aβ peptide fold is common to Aβ fibrils.
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http://dx.doi.org/10.3109/13506129.2016.1146989DOI Listing
June 2016

Enhanced Fibril Fragmentation of N-Terminally Truncated and Pyroglutamyl-Modified Aβ Peptides.

Angew Chem Int Ed Engl 2016 Apr 11;55(16):5081-4. Epub 2016 Mar 11.

Institute for Pharmaceutical Biotechnologie, Ulm University, Helmholtzstrasse 8/1, 89081, Ulm, Germany.

N-terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aβ peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the N terminus of the unmodified peptide protects Aβ fibrils against mechanical stress and fragmentation and explains the high propensity of pE-modified peptides to form small and particularly toxic aggregates.
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http://dx.doi.org/10.1002/anie.201511099DOI Listing
April 2016

Small Molecule Inhibited Parathyroid Hormone Mediated cAMP Response by N-Terminal Peptide Binding.

Sci Rep 2016 Mar 2;6:22533. Epub 2016 Mar 2.

Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, Germany.

Ligand binding to certain classes of G protein coupled receptors (GPCRs) stimulates the rapid synthesis of cAMP through G protein. Human parathyroid hormone (PTH), a member of class B GPCRs, binds to its receptor via its N-terminal domain, thereby activating the pathway to this secondary messenger inside cells. Presently, GPCRs are the target of many pharmaceuticals however, these drugs target only a small fraction of structurally known GPCRs (about 10%). Coordination complexes are gaining interest due to their wide applications in the medicinal field. In the present studies we explored the potential of a coordination complex of Zn(II) and anthracenyl-terpyridine as a modulator of the parathyroid hormone response. Preferential interactions at the N-terminal domain of the peptide hormone were manifested by suppressed cAMP generation inside the cells. These observations contribute a regulatory component to the current GPCR-cAMP paradigm, where not the receptor itself, but the activating hormone is a target. To our knowledge, this is the first report about a coordination complex modulating GPCR activity at the level of deactivating its agonist. Developing such molecules might help in the control of pathogenic PTH function such as hyperparathyroidism, where control of excess hormonal activity is essentially required.
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http://dx.doi.org/10.1038/srep22533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773758PMC
March 2016

The "long tail" of the protein tumbling correlation function: observation by (1)H NMR relaxometry in a wide frequency and concentration range.

J Biomol NMR 2015 Dec 18;63(4):403-415. Epub 2015 Nov 18.

Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, Betty-Heimann-Str. 7, 06120, Halle, Germany.

Inter-protein interactions in solution affect the auto-correlation function of Brownian tumbling not only in terms of a simple increase of the correlation time, they also lead to the appearance of a weak slow component ("long tail") of the correlation function due to a slowly changing local anisotropy of the microenvironment. The conventional protocol of correlation time estimation from the relaxation rate ratio R1/R2 assumes a single-component tumbling correlation function, and thus can provide incorrect results as soon as the "long tail" is of relevance. This effect, however, has been underestimated in many instances. In this work we present a detailed systematic study of the tumbling correlation function of two proteins, lysozyme and bovine serum albumin, at different concentrations and temperatures using proton field-cycling relaxometry combined with R1ρ and R2 measurements. Unlike high-field NMR relaxation methods, these techniques enable a detailed study of dynamics on a time scale longer than the normal protein tumbling correlation time and, thus, a reliable estimate of the parameters of the "long tail". In this work we analyze the concentration dependence of the intensity and correlation time of the slow component and perform simulations of high-field (15)N NMR relaxation data demonstrating the importance of taking the "long tail" in the analysis into account.
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http://dx.doi.org/10.1007/s10858-015-0001-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662726PMC
December 2015

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain.

PLoS One 2015 14;10(9):e0136922. Epub 2015 Sep 14.

Martin-Luther University Halle-Wittenberg, Institute of Physics, Betty-Heimann Str. 7, 06120, Halle, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136922PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569573PMC
June 2016

High-resolution structures of the D-alanyl carrier protein (Dcp) DltC from Bacillus subtilis reveal equivalent conformations of apo- and holo-forms.

FEBS Lett 2015 Aug 17;589(18):2283-9. Epub 2015 Jul 17.

Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.febslet.2015.07.008DOI Listing
August 2015

Novel sulfated phosphoglycolipids from Natronomonas moolapensis.

Chem Phys Lipids 2015 Oct 29;191:8-15. Epub 2015 Jun 29.

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

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 groups.
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http://dx.doi.org/10.1016/j.chemphyslip.2015.06.004DOI Listing
October 2015

Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response.

Proc Natl Acad Sci U S A 2015 May 27;112(19):6230-5. Epub 2015 Apr 27.

Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, D-06120 Halle, Germany; Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06120 Halle, Germany; and Department of Plant Sciences, University of California, Davis, CA 95616

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of auxin/indole-3-acetic acid (AUX/IAA)-inducible repressors, which directly bind to transcriptional auxin response factors (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼ 6.4 μM) were determined by isothermal titration calorimetry. In silico protein-protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein-protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.
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http://dx.doi.org/10.1073/pnas.1424077112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434759PMC
May 2015

NMR-detected brownian dynamics of αB-crystallin over a wide range of concentrations.

Biophys J 2015 Jan;108(1):98-106

Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bpj.2014.11.1858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286604PMC
January 2015