Band-selective universal 90° and 180° rotation pulses covering the aliphatic carbon chemical shift range for triple resonance experiments on 1.2 GHz spectrometers

Biomolecular NMR spectroscopy requires large magnetic field strengths for high spectral resolution. Today’s highest fields comprise proton Larmor frequencies of 1.2 GHz and even larger field strengths are to be expected in the future. In protein triple resonance experiments, various carbon bandwidths need to be excited by selective pulses including the large aliphatic chemical shift range. When the spectrometer field strength is increased, the length of these pulses has to be decreased by the same factor, resulting in higher rf-amplitudes being necessary in order to cover the required frequency region. Currently available band-selective pulses like Q3/Q5 excite a narrow bandwidth compared to the necessary rf-amplitude. Because the maximum rf-power allowed in probeheads is limited, none of the selective universal rotation pulses reported so far is able to cover the full $^{13}$C aliphatic region on 1.2 GHz spectrometers. In this work, we present band-selective 90° and 180° universal rotation pulses (SURBOP90 and SURBOP180) that have a higher ratio of selective bandwidth to maximum rf-amplitude than standard pulses. Simulations show that these pulses perform better than standard pulses, e. g. Q3/Q5, especially when rf-inhomogeneity is taken into account. The theoretical and experimental performance is demonstrated in offset profiles and by implementing the SURBOP pulses in an HNCACB experiment at 1.2 GHz

Combining experiments and simulation to characterise structural and dynamical properties of intrinsically disordered peptides and regions

Intrinsically disordered proteins and regions play important roles in the regulation of protein dynamics and protein-protein interactions. In this thesis two IDPs, both of which have been implicated in neurodegenerative diseases, are explored using fully atomistic molecular dynamics simulations. The first is the N-terminal fragment of the huntingtin protein, which controls the protein’s localisation and function in vivo. The second is the disordered pro domain of the proNGF dimer, which antagonises NGF in the brain. Huntingtin is the causative agent of Huntington’s disease, which is a progres- sive neurodegenerative disease, characterised by CAG repeats in the first exon of Huntingtin, which are translated into a polyglutamine (polyQ) tract, responsible for protein aggregation and subsequent neuron death. Huntingtins poly-Q tract is preceded by a 17-residue regulatory fragment (Htt1-17), which is intrinsically dis- ordered in aqueous environments but forms an amphipathic helix in the presence of TFE or DPC micelles. Htt1-17 regulates localisation and function of the full-length protein and is subject to multiple post-translational modifications in the cell. I used molecular dynamics simulations with a novel enhanced sampling method, to study the effect of phosphorylation, phosphomimetic substitutions and acetylation on the secondary structure of Htt1-19. ProNGF is the precursor to the neurotrophin NGF, and is involved in apoptotic signalling in the brain. A disturbed proNGF:NGF was shown to lead to Alzheimer’s disease. A high-resolution structure of the pro domain has been missing so far. I modelled the proNGF dimer by combining experimental data with long MD simu- lations

Insight into molecular characteristics of SARS-CoV-2 spike protein following D614G point mutation, a molecular dynamics study

Undoubtedly, the SARS-CoV-2 has become a major concern for all societies due to its catastrophic effects on public health. In addition, mutations and changes in the structure of the virus make it difficult to design effective treatment. Moreover, the amino acid sequence of a protein is a major factor in the formation of the second and tertiary structure in a protein. Amino acid replacement can have noticeable effects on the folding of a protein, especially if an asymmetric change (substitution of polar residue with non-polar, charged with an uncharged, positive charge with a negative charge, or large residue with small residue) occurs. D614G as a spike mutant of SARS-CoV-2 previously identified as an associated risk factor with a high mortality rate of this virus. Using structural bioinformatics, our group determined that D614G mutation could cause extensive changes in SARS-CoV-2 behavior including the secondary structure, receptor binding pattern, 3D conformation, and stability of it. Communicated by Ramaswamy H. Sarma. © 2021 Informa UK Limited, trading as Taylor & Francis Group

A computational study of cyclic peptides with vibrational circular dichroism

Cyclic peptides are a class of molecules that has shown antimicrobial potential. These are complex compounds to investigate with their large conformational space and multiple chiral centers. A technique that can be used to investigate both conformational preferences and absolute configuration (AC) is vibrational circular dichroism (VCD). To extract information from the experimental VCD spectra a comparison with calculated spectra is often needed and this is the focus of this thesis: the calculation of VCD spectra. The VCD spectra are very sensitive to small structural changes, and to accurately calculate the spectra, all important conformers need to be identified. The first part of this thesis has been to establish a reliable computational protocol using meta-dynamics to sample the conformational space and ab initio methods to calculate the spectra for cyclic peptides. Using our protocol, we have investigated if VCD alone can determine the AC of cyclic tetra- and hexapeptides. We show that it is possible to determine the AC of the cyclic peptides with two chiral centers while for the peptides with three and four chiral centers, VCD is at best able to reduce the number of possible ACs and further investigation with other techniques is needed. Further, we investigated four cyclic hexapeptides with antimicrobial potential. These peptides, in contrast to the ones used for validating the protocol, consist of several amino acids with long and positively charged side chains. For these peptides, a molecular dynamics based approach provided VCD spectra in better agreement with experiment than our protocol. Reasons for this may be the lack of atomistic detail in the solvent model used during the conformational search and insufficient description of dispersion interactions during the meta-dynamics simulation

Untersuchung von Biopolymereigenschaften mittels Fluoreszenzkorrelationsspektroskopie und computergestützter Methoden

Barsch H. Investigation of biopolymer properties with combined fluorescence correlation spectroscopic and computational methods. Bielefeld (Germany): Bielefeld University; 2008.Neue Erkenntnisse bezüglich der Polymerstruktur von Poly-L-prolinen und einzelsträngigen Polythyminen in wässriger Lösung werden in dieser Arbeit präsentiert. Die Kombination von zeitkorrelierter Einzelphotonenregistrierung (TCSPC) und Fluoreszenzkorrelationsspektroskopie (FCS) von mit Fluoreszenzfarbstoffen markierten Polyprolinderivaten, F-(Pro)N-Trp (mit N = 2 - 10 und F = MR113, MR121, R6G), legen nahe, dass Sub-Populationen der Polyproline mit verringertem Endabstand existieren. Diese Endabstände wurden ebenfalls mittels vereinfachter Molekülmechanik-Simulationen für verschiedene Isomere der Polyproline vorausgesagt. Durch Einbringen von vereinzelten cis-Bindungen in die Prolinpolymere kann gezeigt werden, dass trans-cis-Isomerisierung die Endabstände eines Polymers deutlich verringern kann. Diese Endabstandsverkürzung entspricht den experimentell beobachteten Sub-Populationen, welche bei FCS-Messungen erhöhte Löschung durch photoinduzierten Elektronentransfer ermittelt wurden. Mit einem Fluoreszenzfarbstoff markierte, einzelsträngige Polythymine, MR121-(dT)N (mit N = 2 - 100), wurden durch FCS und moleküldynamische Simulationen untersucht. Die spektroskopischen Messungen liefern längenabhängige hydrodynamische Radien der Polythyminproben, deren Abhängigkeit durch ein Potenzgesetz mit einem Exponenten von 0,5 - 0,7 von der Ionenstärke, I, des Lösungsmittels abhängen. Die Persistenzlänge von MR121-(dT)100, Lp, wurde berechnet und zeigt die Abhängigkeit Lp = I^m mit m = -0,22 ± 0,01. Der Vergleich mit moleküldynamischen Simulationen verschiedener markierter und unmarkierter Polythyminderivate zeigt, dass sich Polythymine wie semiflexible Polmere verhalten, dass für Kettenmoleküle mit einer Länge von N > 30 der Einfluss der Farbstoffmarkierung vernachlässigbar ist und dass elektrostatische Wechselwirkungen bei einer Natriumchlorid-Konzentration von 100 mM vollständig abgeschirmt sind. Weiterhin zeigten die Simulationen, dass die statische Flexibilität der Polythymine durch sterische und geometrische Einschränkungen limitiert ist, was durch eine intrinsische Persistenzlänge von 1,7 nm ausgedrückt werden kann.This thesis presents new insights into the structure of poly-L-proline and single-stranded polythymine polymers in aqueous solution. The combination of time-correlated single-photon counting (TCSPC) and fluorescence correlation spectroscopy (FCS) of fluorescently labeled polyproline derivatives, F-(Pro)N-Trp (with N = 2 - 10 and F = MR113, MR121, R6G), suggest that subpopulations of polyprolines with a reduced end-to-end distance exist. End-to-end distances have also been predicted for different isomers of polyprolines by means of simplified molecular mechanics simulations. Incorporating interspersed cis-bonds into the polyproline shows that trans-cis isomerization leads to significantly shorter end-to-end distances. This fits the experimental results of subpopulations with increased photoinduced electron transfer quenching observed in fluorescent correlation spectroscopy experiments. Fluorescently labeled single-stranded polythymines, MR121-(dT)N (with N = 2 - 100), have been investigated through FCS and molecular dynamics simulations. The spectroscopic measurements resulted in length-dependent hydrodynamic radii of the polythymine probes, which scale according to a power law with an exponent of 0.5 - 0.7 dependent on ionic strength, I, of the solvent. The persistence length of MR121-(dT)100, Lp, has been evaluated which shows a dependence of Lp = I^m with m = - 0.22 ± 0.01. The comparison to molecular dynamics simulations of various labeled and unlabeled polythymine derivatives revealed a semiflexible polymer behavior with neglectable influence of the fluorescent label for polymers with N > 30 and that electrostatic interactions are completely shielded at sodium chloride concentrations of 100 mM. Furthermore, simulation results showed polythymine's static flexibility to be limited by steric and geometric constraints which can be expressed by an intrinsic persistence length of 1.7 nm

Structural and dynamical studies on human epidermal-type fatty acid binding protein using high-resolution NMR spectroscopy

Human epidermal-type fatty acid binding protein (E-FABP) belongs to a family of intracellular non-enzymatic 14-15 kDa lipid binding proteins (LBP) that specifically bind and facilitate the transport of fatty acids, bile acids or retinoids. Their functions have also been associated with fatty acid signalling, cell growth, regulation and differentiation. As a contribution to better understand the structure-function relationship of this protein, the features of its solution structure determined by NMR spectroscopy are reported here. Both unlabeled and 15N-enriched samples of recombinant human E-FABP were used for multidimensional high-resolution NMR. The sequential backbone as well as side-chain resonance assignments have been completed. They are reported here and are also available at the BioMagResBank under the accession number BMRB-5083. The presence of six cysteines in the amino acid sequence of human E-FABP is highly unusual for LBPs. Four of the six cysteines are unique to the E-FABPs: C43, C47, C67 and C87. In the three-dimensional structure of E-FABP, two cysteine pairs (C67/C87 and C120/C127) were identified by X-ray analysis to be close enough to allow disulfide bridge formation, but a S-S bond was actually found only between C120 and C127 [Hohoff et al., 1999]. Since the exclusion of a disulfide bridge between C67 and C87 improved the Rfree factor of the crystallographic model, the existence of a covalent bond between these two side- chains was considered unlikely. This agrees with the NMR data, where SCH resonances have been observed for the cysteine residues C43, C67 (tentative assignment) and C87, thus excluding the possibility of a second disulfide bridge in solution. Based on the NOE and hydrogen exchange data, an ensemble of 20 energy-minimized conformers representing the solution structure of human E-FABP complexed with stearic acid has been obtained. The analysis of homonuclear 2D NOESY and 15N-edited 3D NOESY spectra led to a total of 2926 NOE-derived distance constraints. Furthermore, 37 slow- exchanging backbone amide protons were identified to be part of the hydrogen-bonding network in the >-sheet and subsequently converted into 74 additional distance constraints. Finally, the disulfide bridge between C120 and C127 was defined by 3 upper and 3 lower distance bounds. The structure calculation program DYANA regarded 998 of these constraints as irrelevant, i.e., they did not restrict the distance between two protons. Out of the remaining 2008 non-trivial distance constraints, 371 were intraresidual (i = j), 508 sequential (|i - j| = 1), 233 medium-range (1 4) NOEs. The protein mainly consists of 10 antiparallel -strands forming a >-barrel structure with a large internal cavity. The three-dimensional solution structure of human E-FABP has been determined with a root-mean-square deviation of 0.92 ± 0.11 Å and 1.46 ± 0.10 Å for the backbone and heavy atoms, respectively, excluding the terminal residues. Without the portal region (i.e., for residues 4-26, 40-56, 63-75 and 83-134; the portal region apparently represents the only opening in the protein surface through which the fatty acid ligand can enter and exit the internal binding cavity), an average backbone RMSD of 0.85 ± 0.10 Å was obtained, thus reflecting the higher conformational dispersion in the portal region. Superposition with the X-ray structure of human E-FABP (excluding the terminal residues) yielded average backbone RMSD values of 1.00 ± 0.07 Å for the entire residue range and 0.98 ± 0.06 Å without the portal region. This indicates a close similarity of the crystallographic and the solution structures. The structure coordinates have been deposited at the RCSB data bank under PDB ID code 1JJJ. The measurement of 15N relaxation experiments (T1, T2 and heteronuclear NOE) at three different fields (500, 600 and 800 MHz) provided information on the internal dynamics of the protein backbone. Nearly all non-terminal backbone amide groups showed order parameters S2 > 0.8, with an average value of 0.88 ± 0.04, suggesting a uniformly low backbone mobility in the nanosecond-to-picosecond time range throughout the entire protein sequence. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the >-sheet structure and the conformational exchange (Rex) in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics elaborated here differ from those of the phylogenetically closely related heart-type FABP and the more distantly related ileal lipid binding protein. The results on protein dynamics obtained in this work allow to conclude that the different LBP family members E-FABP, H-FABP and ILBP are characterized by varying stabilities in the protein backbone structures. Hydrogen/deuterium exchange experiments displayed significant differences in the chemical exchange with the solvent for the backbone amide protons belonging to the hydrogen-bonding network in the >-sheets. The >-barrel structure of H- FABP appears to be the most rigid, with exchange processes presumably slower than the millisecond-to-microsecond time range. ILBP, on the other hand, shows the fastest hydrogen exchange as well as a significant number of exchange parameters (Rex), implying a decreased stability in the >-sheet structure. E-FABP, finally, appears to rank between these two proteins based on the hydrogen/deuterium exchange, with Rex terms in the >-strands indicating millisecond-to-microsecond exchange processes like in ILBP

Enhancing the fight against malaria : from genome to structure and activity of a G-protein coupled receptor from the mosquito, Anopheles Gambiae

Includes abstract.Includes bibliographical references (leaves 183-184).G-proton coupled receptors (GPCRs) are excellent drug targets that occupy a central position in the physiology of insects and are involved in transmission of signal from the extracellular to the intracellular side of the cell. Adipokinetic hormone receptors (AKHRs) are GPCRs that mediate physiological functions of the neurohormones, adipokinetic hormones (AKHs) that regulate mobilisation of energy reserves during mosquito flight. Ligand binding to GPCRs depends on the three dimensional (3D) structures of the receptors but to date no crystal structures of insect GPCRs are available. This work focused on building molecular models of AKHR from the genome of the malaria mosquito, identifying its binding site and studying the conformational and structural changes during molecular dynamics of the active and inactive receptor

Structure of HI-6•Sarin-Acetylcholinesterase Determined by X-Ray Crystallography and Molecular Dynamics Simulation: Reactivator Mechanism and Design

Organophosphonates such as isopropyl metylphosphonofluoridate (sarin) are extremely toxic as they phosphonylate the catalytic serine residue of acetylcholinesterase (AChE), an enzyme essential to humans and other species. Design of effective AChE reactivators as antidotes to various organophosphonates requires information on how the reactivators interact with the phosphonylated AChEs. However, such information has not been available hitherto because of three main challenges. First, reactivators are generally flexible in order to change from the ground state to the transition state for reactivation; this flexibility discourages determination of crystal structures of AChE in complex with effective reactivators that are intrinsically disordered. Second, reactivation occurs upon binding of a reactivator to the phosphonylated AChE. Third, the phosphorous conjugate can develop resistance to reactivation. We have identified crystallographic conditions that led to the determination of a crystal structure of the sarinnonaged-conjugated mouse AChE in complex with [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]-oxoazanium dichloride (HI-6) at a resolution of 2.2 Å. In this structure, the carboxyamino-pyridinium ring of HI-6 is sandwiched by Tyr124 and Trp286, however, the oxime-pyridinium ring is disordered. By combining crystallography with microsecond molecular dynamics simulation, we determined the oxime-pyridinium ring structure, which shows that the oxime group of HI-6 can form a hydrogen-bond network to the sarin isopropyl ether oxygen, and a water molecule is able to form a hydrogen bond to the catalytic histidine residue and subsequently deprotonates the oxime for reactivation. These results offer insights into the reactivation mechanism of HI-6 and design of better reactivators

De novo design of a transmembrane Zn²⁺-transporting four-helix bundle.

The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn(2+) and Co(2+), but not Ca(2+), across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn(2+) ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties