Publications by authors named "Vinesh Vijayan"

24 Publications

  • Page 1 of 1

Examining the Transient Dark State in Protein-Quantum Dot Interaction by Relaxation-Based Solution NMR.

J Phys Chem B 2021 Sep 2;125(36):10119-10125. Epub 2021 Sep 2.

School of Chemistry, IISER-Thiruvananthapuram, Maruthamala P.O, Vithura, Thiruvananthapuram, Kerala 695551, India.

We probed the "dark" state involved in the protein-quantum dot (QD) interaction using a relaxation-based solution nuclear magnetic resonance (NMR) approach. We examined the dynamics and exchange kinetics of the ubiquitin-CdTe model system, which undergoes a fast exchange in the transverse relaxation time scale. We applied the recently developed dark-state exchange saturation transfer (DEST), lifetime line broadening (Δ), and exchange-induced chemical shift (δ) solution NMR techniques to obtain a residue-specific binding behavior of the protein on the QD surface. The variation in the estimated N- values clearly shows the dynamic nature of bound Ub. Upon mapping the amino acid residues showing a faster relaxation rate on the electrostatic potential surface of the protein, we have determined that the interaction is preferably electrostatic, and the amino acid residues involved in binding lie on the positively charged surface of the protein. We believe that our experimental approach should provide more in-depth knowledge to engineer new hybrid protein-QD systems in the future.
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http://dx.doi.org/10.1021/acs.jpcb.1c04853DOI Listing
September 2021

Mapping the Fibril Core of the Prion Subdomain of the Mammalian CPEB3 that is Involved in Long Term Memory Retention.

J Mol Biol 2021 07 31;433(15):167084. Epub 2021 May 31.

School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram, Trivandrum 695551, India. Electronic address:

Long-term memory storage is modulated by the prion nature of CPEB3 forming the molecular basis for the maintenance of synaptic facilitation. Here we report that the first prion sub-domain PRD1 of mouse CPEB3 can autonomously form amyloid fibrils in vitro and punctate-like structures in vivo. A ninety-four amino acid sequence within the PRD1 domain, PRD1-core, displays high propensity towards aggregation and associated amyloid characteristics. PRD1-core is characterized using electron microscopy, X-ray diffraction, and solution-state NMR deuterium exchange experiments. Secondary structure elements deduced from solid-state NMR reveal a β-rich core comprising of forty amino acids at the N-terminus of PRD1-core. The synthesized twenty-three amino acid long peptide containing the longest rigid segment (E124-H145) of the PRD1-core rapidly self-aggregates and forms fibrils, indicating a limited aggregation-prone region that could potentially activate the aggregation of the full-length protein. This study provides the first step in identifying the structural trigger for the CPEB3 aggregation process.
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http://dx.doi.org/10.1016/j.jmb.2021.167084DOI Listing
July 2021

Direct Observation of the Self-Aggregation of R3R4 Bi-repeat of Tau Protein.

Chembiochem 2021 Jun 3;22(12):2093-2097. Epub 2021 May 3.

School of Chemistry, IISER Thiruvananthapuram, Maruthamala P.O, Thiruvananthapuram, Kerala, India.

Different cryo-EM derived atomic models of in vivo tau filaments from patients with tauopathies consisted of R3 and R4 repeats of the microtubule-binding domain. In comparison, only the R3 repeat forms the core of the heparin-induced fibrils of the three repeat tau isoforms. For developing therapeutics, it is desirable to have an in vitro tau aggregation system producing fibrils corresponding to the disease morphology. Here we report the self-aggregation of truncated tau segment R3R4 peptide without requiring heparin for aggregation induction. We used NMR spectroscopy and other biophysical methods to monitor the self-aggregation of R3R4. We identified the hexapeptide region in R3 and β-turn region in R4 as the aggregation initiating region of the protein. The solid-state NMR of self-aggregated R3R4 fibrils demonstrated that in addition to R3 residues, residues of R4 were also part of the fibril filaments. The presence of both R3 and R4 residues in the aggregation process and the core of fibril filaments suggest that the aggregation of R3R4 might resemble the in vivo aggregation process.
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http://dx.doi.org/10.1002/cbic.202100013DOI Listing
June 2021

SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer.

Biophys J 2021 05 16;120(10):2019-2029. Epub 2021 Mar 16.

School of Chemistry, Indian Institute of Science Education and Research, Thiruvananthapuram, Kerala, India. Electronic address:

SxIP is a microtubule tip localizing signal found in many +TIP proteins that bind to the hydrophobic cavity of the C-terminal domain of end binding protein 1 (EB1) and then positively regulate the microtubule plus-end tracking of EBs. However, the exact mechanism of microtubule activation of EBs in the presence of SxIP signaling motif is not known. Here, we studied the effect of SxIP peptide on the native conformation of EB1 in solution. Using various NMR experiments, we found that SxIP peptide promoted the dissociation of natively formed EB1 dimer. We also discovered that I224A mutation of EB1 resulted in an unfolded C-terminal domain, which upon binding with the SxIP motif folded to its native structure. Molecular dynamics simulations also confirmed the relative structural stability of EB1 monomer in the SxIP bound state. Residual dipolar couplings and heteronuclear NOE analysis suggested that the binding of SxIP peptide at the C-terminal domain of EB1 decreased the dynamics and conformational flexibility of the N-terminal domain involved in EB1-microtubule interaction. The SxIP-induced disruption of the dimeric interactions in EB1, coupled with the reduction in conformational flexibility of the N-terminal domain of EB1, might facilitate the microtubule association of EB1.
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http://dx.doi.org/10.1016/j.bpj.2021.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204336PMC
May 2021

Residual Dipolar-Coupling-Based Conformational Comparison of Noncovalent Ubiquitin Homodimer with Covalently Linked Diubiquitin.

Chemphyschem 2020 05 7;21(9):888-894. Epub 2020 Apr 7.

School of Chemistry, IISER, Thiruvananthapuram Maruthamala PO, Vithura, Kerala, India.

Although the conformation of the polymer chain of Ubiquitin (Ub) mainly depends on the type of isopeptide linkage connecting two Ub molecules, the non-covalent (noncovalent) interaction between two Ub molecules within the chain could also tune their conformational preference. Here, we studied the conformation of noncovalently formed Ub dimers in solution using residual dipolar couplings (RDCs). Comparing the RDC derived alignment tensor of the noncovalently formed dimer with the two most abundant (K11 and K48) covalent linked Ub dimers revealed that the conformation of K11 linked and noncovalent Ub dimers were similar. Between the various NMR and crystal structures of K11 linked Ub dimers, RDC tensor analysis showed that the structure of K11 linked dimer crystalized at neutral pH is similar to noncovalent dimer. Analogous to the experimental study, the comparison of predicted order matrix of various covalent Ub dimers with that of the experimentally determined order matrix of noncovalent Ub dimer also suggests that the conformation of K11 linked dimers crystalized at neutral pH is similar to the noncovalent dimer.
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http://dx.doi.org/10.1002/cphc.201901100DOI Listing
May 2020

GTP-binding facilitates EB1 recruitment onto microtubules by relieving its auto-inhibition.

Sci Rep 2018 06 28;8(1):9792. Epub 2018 Jun 28.

School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, CET Campus, Thiruvananthapuram, 695016, Kerala, India.

Microtubule plus end-binding protein, EB1 is a key regulator of microtubule dynamics. Auto-inhibitory interaction in EB1 has previously been shown to inhibit its ability to bind to microtubules and regulate microtubule dynamics. However, the factors that promote its microtubule regulatory activity by over-coming the auto-inhibition are less known. Here, we show that GTP plays a critical role in promoting the microtubule-targeting activity of EB1 by suppressing its auto-inhibition. Our biophysical data demonstrate that GTP binds to EB1 at a distinct site in its conserved N-terminal domain. Detailed analyses reveal that GTP-binding suppresses the intra-molecular inhibitory interaction between the globular N-terminus and the C-terminal coiled-coil domain. We further show that mutation of the GTP-binding site residues in N-terminus weakens the affinity for GTP, but also for the C-terminus, indicating overlapping binding sites. Confocal imaging and biochemical analysis reveal that EB1 localization on the microtubules is significantly increased upon mutations of the GTP-binding site residues. The results demonstrate a unique role of GTP in facilitating EB1 interaction with the microtubules by relieving its intra-molecular inhibition. They also implicate that GTP-binding may regulate the functions of EB1 on the cellular microtubules.
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http://dx.doi.org/10.1038/s41598-018-28056-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023887PMC
June 2018

Zn Interrupts R4-R3 Association Leading to Accelerated Aggregation of Tau Protein.

Chemistry 2017 Dec 15;23(67):16976-16979. Epub 2017 Nov 15.

School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram (IISER TVM), Maruthamala P.O, Thiruvananthapuram-695551, Kerala, India.

Direct binding of divalent metal ion, especially Zn , have been shown to increase the rate of tau aggregation and enhance tau toxicity in cells. Hence, understanding the molecular basis of the Zn -accelerated tau aggregation can potentially determine the molecular interactions modulating tau aggregation. Herein, we show that Zn coordinates through the cysteine in R3 repeat and significantly accelerates the aggregation rate of the three repeat tau constructs (K19) but that the coordination is incapable of increasing the aggregation rate of the 20 amino acid peptide derived from the R3 repeat (R3) of tau. The NMR characterization of the binding of Zn to K19, together with the aggregation studies with K19, R3 and R4 peptides, reveal the presence of an aggregation-inhibitory interaction between the R3 and R4 repeat of K19. Our data show that binding of Zn to R3 repeat of tau, weaken the aggregation-inhibiting influence between R3 and R4 repeats, leading to faster aggregation of tau protein.
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http://dx.doi.org/10.1002/chem.201704555DOI Listing
December 2017

Direct Observation of Aggregation-Induced Backbone Conformational Changes in Tau Peptides.

Angew Chem Int Ed Engl 2016 09 11;55(38):11562-6. Epub 2016 Aug 11.

School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram (IISER-TVM), CET campus, Trivandrum-, 695016, India.

In tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion-like fibrils that are capable of propagation. A time-dependent NMR aggregation assay of a slow fibril forming R3-S316P peptide revealed a trans to cis equilibrium shift in the peptide-bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3-S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease.
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http://dx.doi.org/10.1002/anie.201606544DOI Listing
September 2016

Recovery of bulk proton magnetization and sensitivity enhancement in ultrafast magic-angle spinning solid-state NMR.

J Phys Chem B 2015 Feb 3;119(7):2908-20. Epub 2015 Feb 3.

Department of NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry , 37077 Göttingen, Germany.

The sensitivity of solid-state NMR experiments is limited by the proton magnetization recovery delay and by the duty cycle of the instrument. Ultrafast magic-angle spinning (MAS) can improve the duty cycle by employing experiments with low-power radio frequency (RF) irradiation which reduce RF heating. On the other hand, schemes to reduce the magnetization recovery delay have been proposed for low MAS rates, but the enhancements rely on selective transfers where the bulk of the (1)H magnetization pool does not contribute to the transfer. We demonstrate here that significant sensitivity enhancements for selective and broadband experiments are obtained at ultrafast MAS by preservation and recovery of bulk (1)H magnetization. We used [(13)C, (15)N]-labeled glutamine as a model compound, spinning in a 1.3 mm rotor at a MAS frequency of 65 kHz. Using low-power (1)H RF (13.4 kHz), we obtain efficient (1)H spin locking and (1)H-(13)C decoupling at ultrafast MAS. As a result, large amounts of (1)H magnetization, from 35% to 42% of the initial polarization, are preserved after cross-polarization and decoupling. Restoring this magnetization to the longitudinal axis using a flip-back pulse leads to an enhancement of the sensitivity, an increase ranging from 14% to 21% in the maximal achievable sensitivity regime and from 24% to 50% in the fast pulsing regime, and to a shortening of the optimal recycling delay to 68% of its original duration. The analysis of the recovery and sensitivity curves reveals that the sensitivity gains do not rely on a selective transfer where few protons contribute but rather on careful conservation of bulk (1)H magnetization. This makes our method compatible with broadband experiments and uniformly labeled materials, in contrast to the enhancement schemes proposed for low MAS. We tested seven different cross-polarization schemes and determined that recovery of bulk (1)H magnetization is a general method for sensitivity enhancement. The physical insight gained about the behavior of proton magnetization sharing under spin lock will be helpful to break further sensitivity boundaries, when even higher external magnetic fields and faster spinning rates are employed.
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http://dx.doi.org/10.1021/jp511987yDOI Listing
February 2015

A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme.

J Magn Reson 2013 Mar 4;228:45-9. Epub 2013 Jan 4.

Max Planck Institute for Biophysical Chemistry, Department of NMR-based Structural Biology, Am Fassberg 11, 37077 Göttingen, Germany.

The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-(13)C]Glucose ([2-(13)C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-(13)C]pyruvate produced mainly by glycolysis and [3-(13)C]/[1,3-(13)C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs (13)Cβ-(13)Cγ2 and (13)Cα-(13)Cγ1 in Val, and (13)Cγ-(13)Cδ2 and (13)Cβ-(13)Cδ1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin.
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http://dx.doi.org/10.1016/j.jmr.2012.12.017DOI Listing
March 2013

β-Sheet core of tau paired helical filaments revealed by solid-state NMR.

J Am Chem Soc 2012 Aug 15;134(34):13982-9. Epub 2012 Aug 15.

NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

One of the hallmarks of Alzheimer's disease is the self-assembly of the microtubule-associated protein tau into fibers termed "paired helical filaments" (PHFs). However, the structural basis of PHF assembly at atomic detail is largely unknown. Here, we applied solid-state nuclear magnetic resonance (ssNMR) spectroscopy to investigate in vitro assembled PHFs from a truncated three-repeat tau isoform (K19) that represents the core of PHFs. We found that the rigid core of the fibrils is formed by amino acids V306 to S324, only 18 out of 99 residues, and comprises three β-strands connected by two short kinks. The first β-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [(15)N:(13)C]-labeled K19 fibrils show that β-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the β-sheet structure, and polymorphism in ssNMR spectra is observed. In particular, residues K321-S324 exhibit two sets of resonances. Experiments on K19 C322A PHFs further confirm the influence of disulfide bond formation on the core structure. Our structural data are supported by H/D exchange NMR measurements on K19 as well as a truncated four-repeat isoform of tau (K18). Site-directed mutagenesis studies show that single-point mutations within the three different β-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the β-structure-rich regions for tau aggregation.
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http://dx.doi.org/10.1021/ja305470pDOI Listing
August 2012

Tailored low-power cross-polarization under fast magic-angle spinning.

J Magn Reson 2010 Aug 6;205(2):216-23. Epub 2010 May 6.

Max Planck Institute for Biophysical Chemistry, Solid-state NMR, Göttingen 37077, Germany.

High static magnetic fields and very fast magic-angle spinning (MAS) promise to improve resolution and sensitivity of solid-state NMR experiments. The fast MAS regime has permitted the development of low-power cross-polarization schemes, such as second-order cross-polarization (SOCP), which prevent heat deposition in the sample. Those schemes are however limited in bandwidth, as weak radio-frequency (RF) fields only cover a small chemical shift range for rare nuclei (e.g. (13)C). Another consideration is that the efficiency of cross-polarization is very sensitive to magnetization decay that occurs during the spin-lock pulse on the abundant nuclei (e.g. (1)H). Having characterized this decay in glutamine at 60 kHz MAS, we propose two complementary strategies to tailor cross-polarization to desired spectral regions at low RF power. In the case of multiple sites with small chemical shift dispersion, a larger bandwidth for SOCP is obtained by slightly increasing the RF power while avoiding recoupling conditions that lead to fast spin-lock decay. In the case of two spectral regions with large chemical shift offset, an extension of the existing low-power schemes, called MOD-CP, is introduced. It consists of a spin-lock on (1)H and an amplitude-modulated spin-lock on the rare nucleus. The range of excited chemical shifts is assessed by experimental excitation profiles and numerical simulation of an I(2)S spin system. All SOCP-based schemes exhibit higher sensitivity than high-power CP schemes, as demonstrated on solid (glutamine) and semi-solid (hydrated, micro-crystalline ubiquitin) samples.
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http://dx.doi.org/10.1016/j.jmr.2010.04.019DOI Listing
August 2010

Low-power solid-state NMR experiments for resonance assignment under fast magic-angle spinning.

Chemphyschem 2009 Sep;10(13):2205-8

NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

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http://dx.doi.org/10.1002/cphc.200900439DOI Listing
September 2009

Plasticity of the PAS domain and a potential role for signal transduction in the histidine kinase DcuS.

Nat Struct Mol Biol 2008 Oct 28;15(10):1031-9. Epub 2008 Sep 28.

Max-Planck-Institute for Biophysical Chemistry, Department of NMR-Based Structural Biology, Am Fassberg 11, 37077 Göttingen, Germany.

The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solid-state NMR, structural modeling and mutagenesis. Experimental data of an individual cytoplasmic Per-Arnt-Sim (PAS) domain were compared to structural models generated in silico. These studies, together with previous NMR work on the periplasmic PAS domain, enabled structural investigations of a membrane-embedded 40-kDa construct by solid-state NMR, comprising both PAS segments and the membrane domain. Structural alterations are largely limited to protein regions close to the transmembrane segment. Data from isolated and multidomain constructs favor a disordered N-terminal helix in the cytoplasmic domain. Mutations of residues in this region strongly influence function, suggesting that protein flexibility is related to signal transduction toward the kinase domain and regulation of kinase activity.
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http://dx.doi.org/10.1038/nsmb.1493DOI Listing
October 2008

High-resolution 3D structure determination of kaliotoxin by solid-state NMR spectroscopy.

PLoS One 2008 Jun 4;3(6):e2359. Epub 2008 Jun 4.

Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002359PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2387072PMC
June 2008

A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA.

J Mol Biol 2008 Mar 16;377(2):512-23. Epub 2008 Jan 16.

Department of Structural Chemistry, University of Göttingen, Tammannstrasse 4, 37077 Göttingen, Germany.

Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metabolism of citrate in response to its extracellular concentration. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor.
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http://dx.doi.org/10.1016/j.jmb.2008.01.024DOI Listing
March 2008

Fast high-resolution protein structure determination by using unassigned NMR data.

Angew Chem Int Ed Engl 2007 ;46(7):1176-9

Department of NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

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http://dx.doi.org/10.1002/anie.200603213DOI Listing
April 2007

1H, 15N, and 13C resonance assignment of the C2A domain of rabphilin 3A.

J Biomol NMR 2006 ;36 Suppl 1:20

Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany.

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http://dx.doi.org/10.1007/s10858-005-6051-zDOI Listing
August 2007

Simultaneous measurement of protein one-bond residual dipolar couplings without increased resonance overlap.

J Magn Reson 2005 Jun;174(2):245-53

Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

A NMR strategy designed to measure simultaneously and without increased resonance overlap scalar and dipolar couplings (RDCs) in (13)C-, (15)N-labeled proteins is presented. Contrary to common schemes for simultaneous measurement of RDCs, a single reference experiment is used for the extraction of more than one type of coupling, thereby reducing the required measurement time. This is accomplished by a common reference spectrum followed by a series of interleaved experiments, in which a particular coupling dependent parameter is varied according to the quantitative J-correlation method or using accordion spectroscopy. To illustrate this idea, we have modified the 3D TROSY-HNCO and the 3D CBCA(CO)NH experiment allowing efficient measurement of one-bond (1)D(NH), (1)D(C'N), (1)D(CalphaHalpha), (1)D(CbetaHbeta), and (1)D(CalphaC') couplings in small to medium sized proteins. In addition, the experiments are expected to be useful for largely unfolded proteins, which show strong resonance overlap but have very favorable relaxation properties. Measurement of RDCs is demonstrated on uniformly (15)N-(13)C-labeled ubiquitin and on the sensory domain of the membraneous two-component fumarate sensor DcuS of Escherichia coli (17 kDa). DcuS was found to be unstable and to precipitate in one to two weeks. RDCs obtained from these experiments are in good agreement with the 1.8A X-ray structure of ubiquitin.
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http://dx.doi.org/10.1016/j.jmr.2005.02.010DOI Listing
June 2005

Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization.

J Biol Chem 2005 Jun 15;280(25):23766-70. Epub 2005 Apr 15.

Department for NMR-based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.
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http://dx.doi.org/10.1074/jbc.C500064200DOI Listing
June 2005

The nature of the stimulus and of the fumarate binding site of the fumarate sensor DcuS of Escherichia coli.

J Biol Chem 2005 May 21;280(21):20596-603. Epub 2005 Mar 21.

Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, 55099 Mainz, Germany.

DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C(4) -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C(4)-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 A apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mm for the C(4)-dicarboxylates and 0.5 mm for 3-nitropropionate or d-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg(107)-X(2)-His(110)-X(9)-Phe(120)-X(26)-Arg(147)-X-Phe(149)) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or d-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C(4)-dicarboylate binding domain. The amino acid residues responding to fumarate or d-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C(4)-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.
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http://dx.doi.org/10.1074/jbc.M502015200DOI Listing
May 2005

Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis.

EMBO J 2004 Sep 9;23(18):3632-42. Epub 2004 Sep 9.

Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram-positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix-turn-helix DNA-binding motif as part of a five-helix bundle that is extended by an antiparallel beta-sheet. We show that CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript.
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http://dx.doi.org/10.1038/sj.emboj.7600367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC517608PMC
September 2004

The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli.

J Biol Chem 2003 Oct 7;278(40):39185-8. Epub 2003 Aug 7.

Max Planck Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany.

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.
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http://dx.doi.org/10.1074/jbc.C300344200DOI Listing
October 2003
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