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Atomic details of structured water molecules are indispensable to understand the thermodynamics of important biological processes including the proton conduction mechanism of the M2 protein. Despite the expectation of structured water molecules based on crystal structures of Influenza A M2, only two water populations have been observed by NMR in reconstituted lipid bilayer samples. These are the bulk‐ and lipid‐associated water populations typically seen in membrane samples. Here, we detect a bound water molecule at a chemical shift of 11 ppm, located near the functional histidine 37 residue in the M2 conductance domain, which comprises residues 18 to 60. Combining 100 kHz magic‐angle spinning NMR, dynamic nuclear polarization and density functional theory calculations, we show that the bound water forms a hydrogen bond to the δ1 nitrogen of histidine 37., Water structure impacts biological processes. Within the Influenza A M2 proton channel, a bound water molecule proton was detected at a chemical shift of 11 ppm. Combining magic‐angle spinning NMR, dynamic nuclear polarization and quantum chemical calculations, the water was found hydrogen bonded to Nδ1 of the functional histidine 37 residue.

Phase separation is a fundamental physicochemical process underlying the spatial arrangement and coordination of cellular events. Detailed characterization of biomolecular phase separation requires experimental access to the internal environment of dilute and especially condensed phases at high resolution. In this study, we take advantage from the ubiquitous presence of sodium ions in biomolecular samples and present the potentials of 23 Na NMR as a proxy to report the internal fluidity of biomolecular condensed phases. After establishing the temperature and viscosity dependence of 23 Na NMR relaxation rates and translational diffusion coefficient, we demonstrate that 23 Na NMR probes of rotational and translational mobility of sodium ions are capable of capturing the increasing levels of confinement in agarose gels in dependence of agarose concentration. The 23 Na NMR approach is then applied to a gel-forming FG peptide, part of the nuclear pore complex involved in controlling the traffic between cytoplasm and cell nucleus. It is shown that the 23 Na NMR together with the 17 O NMR provide a detailed picture of the sodium ion and water mobility within the interior of the FG peptide hydrogel. As another example, we study phase separation in water-triethylamine mixture and provide evidence for the presence of multiple microscopic environments within the triethylamine-rich phase. Our results highlight the potentials of 23 Na NMR in combination with 17 O NMR in studying biological phase separation, in particular with regards to the molecular properties of biomolecular condensates and their regulation through various physico- and biochemical factors. This article is protected by copyright. All rights reserved.

A contribution of α-Synuclein (α-Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related β-Synuclein (β-Syn) on these disorders remains enigmatic. β-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD β-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of β-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced β-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with β-Syn, thereby enabling structural and functional modifications. Interaction of DCH with β-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric β-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and β-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that β-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.

Peptidylprolyl isomerases (PPIases) catalyze cis/trans isomerization of prolines. The PPIase CypA colocalizes with the Parkinson's disease (PD)‐associated protein α‐synuclein in cells and interacts with α‐synuclein oligomers. Herein, we describe atomic insights into the molecular details of the α‐synuclein/CypA interaction. NMR spectroscopy shows that CypA catalyzes isomerization of proline 128 in the C‐terminal domain of α‐synuclein. Strikingly, we reveal a second CypA‐binding site formed by the hydrophobic sequence 47GVVHGVATVA56, termed PreNAC. The 1.38 Å crystal structure of the CypA/PreNAC complex displays a contact between alanine 53 of α‐synuclein and glutamine 111 in the catalytic pocket of CypA. Mutation of alanine 53 to glutamate, as found in patients with early‐onset PD, weakens the interaction of α‐synuclein with CypA. Our study provides high‐resolution insights into the structure of the PD‐associated protein α‐synuclein in complex with the most abundant cellular cyclophilin., The high‐resolution structure of the PreNAC region of α‐synuclein in complex with the peptidylprolyl isomerase cyclophilin A provides a novel entry point for the modulation of α‐synuclein‐related neurotoxicity. The A53E mutation of α‐synuclein, which causes early‐onset Parkinson's disease, weakens the interaction of α‐synuclein with cyclophilin A.

In the root endophyte Serendipita indica, several lectin-like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in β-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long-chain β1-3-glucan but has no affinity for shorter β1-3- or β1-6-linked glucose oligomers. Comparative analysis with the previously identified β-glucan-binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require β1-6-linked glucose for efficient binding to branched β1-3-glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two β-glucan-binding lectins. Our results highlight the importance of the β-glucan cell wall component in plant-fungus interactions and the potential of β-glucan-binding lectins as specific detection tools for fungi in vivo.

Background The SNCA gene encoding α-synuclein (αSyn) is the first gene identified to cause autosomal-dominant Parkinson's disease (PD). Objective We report the identification of a novel heterozygous A30G mutation of the SNCA gene in familial PD and describe clinical features of affected patients, genetic findings, and functional consequences. Methods Whole exome sequencing was performed in the discovery family proband. Restriction digestion with Bbvl was used to screen SNCA A30G in two validation cohorts. The Greek cohort included 177 familial PD probands, 109 sporadic PD cases, and 377 neurologically healthy controls. The German cohort included 136 familial PD probands, 380 sporadic PD cases, and 116 neurologically healthy controls. We also conducted haplotype analysis using 13 common single nucleotide variants around A30G to determine the possibility of a founder effect for A30G. We then used biophysical methods to characterize A30G αSyn. Results We identified a novel SNCA A30G (GRCh37, Chr4:90756730, c.89 C>G) mutation that co-segregated with the disease in five affected individuals of three Greek families and was absent from controls. A founder effect was strongly suggested by haplotype analysis. The A30G mutation had a local effect on the intrinsically disordered structure of αSyn, slightly perturbed membrane binding, and promoted fibril formation. Conclusion Based on the identification of A30G co-segregating with the disease in three families, the absence of the mutation in controls and population databases, and the observed functional effects, we propose SNCA A30G as a novel causative mutation for familial PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

15N-Spinrelaxationsraten konnen wichtige Informationen uber Fernstruktur und interne Bewegungen von Membranproteinen bereitstellen. Kombiniert mit einem verbesserten Berechnungsprotokoll offenbart die mittels Relaxationsraten abgeleitete Struktur des 283 Reste umfassenden humanen spannungsabhangigen Anionenkanals ein anisotrop geformtes Fass mit einer fest verankerten N-terminalen Helix. Diese Arbeit eroffnet somit einen NMR-spektroskopischen Ansatz, um die Struktur und Dynamik von Membranproteinen der Saugetiere mit hoher Exaktheit und Auflosung zu bestimmen.

Protein dynamics occurring on a wide range of timescales play a crucial role in governing protein function. Particularly, motions between the globular rotational correlation time (τc ) and 40 μs (supra-τc window), strongly influence molecular recognition. This supra-τc window was previously hidden, owing to a lack of experimental methods. Recently, we have developed a high-power relaxation dispersion (RD) experiment for measuring kinetics as fast as 4 μs. For the first time, this method, performed under super-cooled conditions, enabled us to detect a global motion in the first β-turn of the third IgG-binding domain of protein G (GB3), which was extrapolated to 371±115 ns at 310 K. Furthermore, the same residues show the plasticity in the model-free residual dipolar coupling (RDC) order parameters and in an ensemble encoding the supra-τc dynamics. This β-turn is involved in antibody binding, exhibiting the potential link of the observed supra-τc motion with molecular recognition.

The translocator protein (TSPO) is an integral membrane protein that interacts with a wide variety of endogenous ligands, such as cholesterol and porphyrins, and is also the target for several small molecules with substantial in vivo efficacy. When complexed with the TSPO-specific radioligand (R)-PK11195, TSPO folds into a rigid five-helix bundle. However, little is known about the structure and dynamics of TSPO in the absence of high-affinity ligands. By means of NMR spectroscopy, we show that TSPO exchanges between multiple conformations in the absence of (R)-PK11195. Extensive motions on time scales from pico- to microseconds occur all along the primary sequence of the protein, leading to a loss of stable tertiary interactions and local unfolding of the helical structure in the vicinity of the ligand-binding site. The flexible nature of TSPO highlights the importance of conformational plasticity in integral membrane proteins.

Motions play a vital role in the functions of many proteins. Discrete conformational transitions to excited states, happening on timescales of hundreds of microseconds, have been extensively characterized. On the other hand, the dynamics of the ground state are widely unexplored. Newly developed high-power relaxation dispersion experiments allow the detection of motions up to a one-digit microsecond timescale. These experiments showed that side chains in the hydrophobic core as well as at protein-protein interaction surfaces of both ubiquitin and the third immunoglobulin binding domain of protein G move on the microsecond timescale. Both proteins exhibit plasticity to this microsecond motion through redistribution of the populations of their side-chain rotamers, which interconvert on the picosecond to nanosecond timescale, making it likely that this "population shuffling" process is a general mechanism.

Paramagnetische Effekte ermoglichen einzigartige Einblicke in die Struktur und Dynamik von Biomolekulen. Um diese nutzbar zu machen, wurde eine Methode entwickelt, in welcher ein Lanthanoid-Tag an ein Reporterprotein angebracht wird, das anschliesend an das Zielprotein bindet und dadurch paramagnetische Effekte im Zielprotein hervorruft. Dieser Ansatz bietet einen Zugang zu einer grosen Zahl paramagnetischer Effekte auf der Basis unabhangiger Molekulausrichtungen in hochmolekularen Proteinen unbekannter 3D-Struktur.

A conserved family of tryptophan-rich sensory proteins (TspO) mediates the transport of heme degradation intermediates across membranes. In eukaryotes, the homologous mitochondrial translocator protein (TSPO) binds cholesterol and radioligands as monomer. On the basis of the mammalian TSPO structure, bioinformatic analysis, and a 10 A resolution electron microscopy map of TspO from Rhodobacter sphaeroides, we developed a model of the tertiary and quaternary structure of TspO that is in agreement with available mutagenesis data. Our study provides insight into the conformational basis for the restricted interaction of bacterial TspO with radioligands and the functional oligomerization state of bacterial TspO proteins.

Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment.

α-Synuclein (aS), a small protein containing 140 amino acids, undergoes self-assembly into amyloid fibrils and plaques (Lewy bodies), which are pathological hallmarks of Parkinson’s disease (PD) as well as other neurodegenerative diseases.[1] While oligomeric species of aS are considered to exert the neurotoxic activity[2] that can be rescued by reducing those oligomers either by diversion to smaller oligomers[3] or acceleration of fibril formation, cell-to-cell transmission in nontransgenic mice points to a direct role of the fibrils in spreading the disease from peripheral to central neurons.[4] Understanding the molecular interactions that lead to misfolding strongly relies on the availability of suited biophysical methods that can access the structure of these states. For the monomeric form, magnetic resonance techniques gave evidence for a natively disordered yet partially folded protein[5a,b] that upon binding to lipid vesicles adopts an α-helical structure.[6–8] When aS is aggregated into fibrils, X-ray and electron diffraction studies[9a,b] revealed that it arranges in a classical cross-β conformation, where the individual β-sheets arrange perpendicularly to the fibril axis with spacing of 4.7–4.8 A along the fibril axis and 10–11 A perpendicular to the axis. In particular, aS stacks in a parallel, in-register arrangement as revealed by continuous-wave (CW) electron paramagnetic resonance (EPR)[10] and more recently supported by solid-state nuclear magnetic resonance (ss-NMR) spectroscopy.[11]

Objective Whereas the contribution of α-synuclein to neurodegeneration in Parkinson disease is well accepted, the putative impact of its close homologue, β-synuclein, is enigmatic. β-Synuclein is widely expressed throughout the central nervous system, as is α-synuclein, but the physiological functions of both proteins remain unknown. Recent findings have supported the view that β-synuclein can act as an ameliorating regulator of α-synuclein–induced neurotoxicity, having neuroprotective rather than neurodegenerative capabilities, and being nonaggregating due to the absence of most of the aggregation-promoting NAC domain. However, a mutation of β-synuclein linked to dementia with Lewy bodies rendered the protein neurotoxic in transgenic mice, and fibrillation of β-synuclein has been demonstrated in vitro. Methods Neurotoxicity and aggregation properties of α-, β-, and γ-synuclein were comparatively elucidated in the rat nigro-striatal projection and in cultured neurons. Results Supporting the hypothesis that β-synuclein can act as a neurodegeneration-inducing factor, we demonstrated that wild-type β-synuclein is neurotoxic for cultured primary neurons. Furthermore, β-synuclein formed proteinase K–resistant aggregates in dopaminergic neurons in vivo, leading to pronounced and progressive neurodegeneration in rats. Expression of β-synuclein caused mitochondrial fragmentation, but this fragmentation did not render mitochondria nonfunctional in terms of ion handling and respiration even at late stages of neurodegeneration. A comparison of the neurodegenerative effects induced by α-, β-, and γ-synuclein revealed that β-synuclein was eventually as neurotoxic as α-synuclein for nigral dopaminergic neurons, whereas γ-synuclein proved to be nontoxic and had very low aggregation propensity. Interpretation Our results suggest that the role of β-synuclein as a putative modulator of neuropathology in aggregopathies like Parkinson disease and dementia with Lewy bodies needs to be revisited. Ann Neurol 2013;74:109–118

Purpose: A novel unconditionally stable, explicit numerical method is introduced to the field of modelingbraincancer progression on a tissue level together with an inverse problem (IP) based on optimal control theory that allows for automated modelcalibration with respect to observations in clinical imaging data. Methods: Biophysical models of cancer progression on a tissue level are in general based on the assumption that the spatiotemporal spread of cancerous cells is determined by cell division and net migration. These processes are typically described in terms of a parabolic partial differential equation (PDE). In the present work a parallelized implementation of an unconditionally stable, explicit Euler (EE⋆) time integration method for the solution of this PDE is detailed. The key idea of the discussed EE⋆ method is to relax the strong stability requirement on the spectral radius of the coefficient matrix by introducing a subdivision regime for a given outer time step. The performance is related to common implicit numerical methods. To quantify the numerical error, a simplified model that has a closed form solution is considered. To allow for a systematic, phenomenological validation a novel approach for automated modelcalibration on the basis of observations in medical imaging data is developed. The resulting IP is based on optimal control theory and manifests as a large scale, PDE constrained optimization problem. Results: The numerical error of the EE⋆ method is at the order of standard implicit numerical methods. The computing times are well below those obtained for implicit methods and by that demonstrate efficiency. Qualitative and quantitative analysis in 12 patients demonstrates that the obtained results are in strong agreement with observations in medical imaging data. Rating simulation success in terms of the mean overlap between model predictions and manual expert segmentations yields a success rate of 75% (9 out of 12 patients). Conclusions: The discussed EE⋆ method provides desirable features for image-basedmodelcalibration or hybrid image registration algorithms in which the model serves as a biophysical prior. This is due to (i) ease of implementation, (ii) low memory requirements, (iii) efficiency, (iv) a straightforward interface for parameter updates, and (v) the fact that the method is inherently matrix-free. The explicit time integration method is confirmed via experiments for automated modelcalibration. Qualitative and quantitative analysis demonstrates that the proposed framework allows for recovering observations in medical imaging data and by that phenomenological model validity.

Protein-mediated polarization transfer: Ligands L1 and L2 that competitively bind to a protein are subject to indirect spin-polarization transfer through the binding site of the protein. If protons HL1 of one ligand are hyperpolarized by dynamic nuclear polarization (DNP, see picture), signal intensities in the NMR spectrum of the second ligand become enhanced. The relative build-up of signal of the second ligand yields information on its binding epitope.

Physiological processes such as protein folding and molecular recognition are intricately linked to their dynamic signature, which is reflected in their thermal coefficient. In addition, the local conformational entropy is directly related to the degrees of freedom, which each residue possesses within its conformational space. Therefore, the temperature dependence of the local conformational entropy may provide insight into understanding how local dynamics may affect the stability of proteins. Here, we analyze the temperature dependence of internal methyl group dynamics derived from the cross-correlated relaxation between dipolar couplings of two CH bonds within ubiquitin. Spanning a temperature range from 275 to 308 K, internal methyl group dynamics tend to increase with increasing temperature, which translates to a general increase in local conformational entropy. With this data measured over multiple temperatures, the thermal coefficient of the methyl group order parameter, the characteristic thermal coefficient, and the local heat capacity were obtained. By analyzing the distribution of methyl group thermal coefficients within ubiquitin, we found that the N-terminal region has relatively high thermostability. These results indicate that methyl groups contribute quite appreciably to the total heat capacity of ubiquitin through the regulation of local conformational entropy.

Complex problem: The crystal structure of p38α mitogen-activated protein kinase in complex with a dibenzo[a,d]cycloheptenone inhibitor was found to be incompatible with NMR data of the same complex in solution. By using inferential structure determination (ISD) with restraints from X-ray crystallography and NMR spectra, a structure that is compatible with both data sets and very close to the X-ray crystal structure was generated (see picture).

Molecular recognition plays a central role in many biological processes. For enzymatic reactions and slow protein–protein recognition events, turn-over rates and on-rates in the millisecond-to-second time scale have been connected to internal protein dynamics detected with atomic resolution by NMR spectroscopy, and in particular conformational sampling could be established as a mechanism for enzyme–substrate and protein–protein recognition. Recent theoretical studies indicate that faster rates of conformational interconversion in the microsecond time scale might limit on-rates for protein–protein recognition. However experimental proofs were lacking so far, mainly because such rates could not be determined accurately enough and kinetic experiments in the microsecond time range are difficult to perform. Nevertheless, for proteins and TAR-RNA, recent studies based on residual dipolar couplings (RDCs) and other NMR spectroscopy techniques have detected substantial internal dynamics in a time window from the rotational correlation time tc (one-digit nanoseconds) to approximately 50 ms, called the supra-tc window in the following. However, the exact rates of internal dynamics within this four orders of magnitude wide time window could not be determined. Supra-tc dynamics in ubiquitin [9] and TAR-RNA could be connected to the conformational sampling required for molecular recognition. While the amplitudes of motions have been indirectly detected by RDCs and characterized in great detail, it has so far been impossible to directly observe these motions and to determine the exact rate of these supra-tc motions. In contrast, conformational sampling in enzymes occurs on a time scale that is 100 to 1000 times slower than supra-tc dynamics and therefore NMR relaxation dispersion (RD) techniques have been able to establish the functional link to enzyme kinetics with atomic resolution at physiological conditions. 5] However, for technical reasons, RD is not sensitive to motion faster than approximately 50 ms (RD window) and therefore does not access motion in the supra-tc window at room temperature. Here we determine the rate of interconversion between conformers of free ubiquitin by a combination of NMR RD experiments in super-cooled solution and dielectric relaxation spectroscopy (DR). Furthermore, we corroborate the motional amplitudes in the RDC-derived ensembles quantitatively with the observed amplitudes of RD and DR. The methods utilized herein can be used to directly study protein dynamics in a time range that was previously inaccessible. Significant motional amplitude in the supra-tc window has been observed using RDC measurements, and was connected to the conformational sampling for a protein in the ground

The major component of neural inclusions that are the pathological hallmark of Parkinson's disease are amyloid fibrils of the protein α-synuclein (aS). Here we investigated if the disease-related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two-dimensional proton-detected high-resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of β-strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano-second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation-induced change in the conformation of aS aggregates.

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N-terminal, soluble domain in the intermembrane space (IMS) and a C-terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N-terminal domain of Tim23, Tim23N (residues 1-96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71-84. In a charge-hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network.

Ca(2+) signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca(2+)-CaM binds a conserved region in the priming proteins Munc13-1 and ubMunc13-2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca(2+) signals. We solved the structure of Ca(2+)(4)-CaM in complex with the CaM-binding domain of Munc13-1, which features a novel 1-5-8-26 CaM-binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13-2 isoform. The N-module can be dissociated with EGTA to form the half-loaded Munc13/Ca(2+)(2)-CaM complex. The Ca(2+) regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca(2+)-CaM interactions, where the C-module provides a high-affinity interaction activated at nanomolar [Ca(2+)](i), whereas the N-module acts as a sensor at micromolar [Ca(2+)](i). This Ca(2+)/CaM-binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca(2+)-dependent modulation of short-term synaptic plasticity.

The relation of α-synuclein (αS) aggregation to Parkinson's disease has long been recognized, but the pathogenic species and its molecular properties have yet to be identified. To obtain insight into the properties of αS in an aggregation-prone state, we studied the structural properties of αS at acidic pH using NMR spectroscopy and computation. NMR demonstrated that αS remains natively unfolded at lower pH, but secondary structure propensities were changed in proximity to acidic residues. The ensemble of conformations of αS at acidic pH is characterized by a rigidification and compaction of the Asp and Glu-rich C-terminal region, an increased probability for proximity between the NAC-region and the C-terminal region and a lower probability for interactions between the N- and C-terminal regions.

Summer rainfall in the Yangtze River basin is predicted using neural network techniques. Input variables (predictors) for the neural network are the Southern Oscillation Index (SOI), the East Atlantic/Western Russia (EA/WR) pattern, the Scandinavia (SCA) pattern, the Polar/Eurasia (POL) pattern and several indices calculated from sea surface temperatures (SST), sea level pressures (SLP) and snow data from December to April for the period from 1993 to 2002. The output variable of the neural network is rainfall from May to September for the period from 1994 to 2002, which was previously classified into six different regions by means of a principal component analysis (PCA). Rainfall is predicted from May to September 2002. The winter SST and SLP indices are identified to be the most important predictors of summer rainfall in the Yangtze River basin. The Tibetan Plateau snow depth, the SOI and the other teleconnection indices seem to be of minor importance for an accurate prediction. This may be the result of the length of the available time series, which does not allow a deeper analysis of the impact of multi-annual oscillations. The neural network algorithms proved to be capable of explaining most of the rainfall variability in the Yangtze River basin. For five out of six regions, our predictions explain at least 77% of the total variance of the measured rainfall. Copyright © 2007 Royal Meteorological Society

Daily precipitation data of 148 weather stations located in the Yangtze River basin (P.R. China) are analysed to detect cycles in the annual frequency of occurrence of precipitation events of 1-, 5- and 10 days duration. These events were defined in terms of exceedances of some selected thresholds. The events corresponding to 10, 25 and 30 mm thresholds for 1-, 5- and 10-day precipitation totals, respectively, are analysed in detail. For the identification of cycles, basin-wide averaged standardized time series of frequency of precipitation events are used. It is found that peaks in the smoothed time series occurred around 1974, 1982 and 1991. The Fourier, autoregressive and wavelet analyses reveal distinct cycles of 7–9 and 2–3 year periods, which dominate large parts of the time series. In addition, a shift towards a 4–5 year period in the annual frequency of precipitation events is noticed since the mid- to late-nineties. Major peaks in the annual frequency of occurrence of precipitation events are expected to occur around 2012, 2015 and 2018 according to the spectrum analyses. Copyright © 2007 Royal Meteorological Society

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D-chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC-CC, MALDI-TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of 13C', 13Calpha, and 13Cbeta resonance frequencies suggests that 13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR.

Complex polymers are distributed in more than one direction of molecular heterogeneity. In addition to the molar mass distribution, they are frequently distributed with respect to chemical composition, functionality, and molecular heterogeneity. One approach for the analysis of the heterogeneity of complex polymers is their chromatographic separation by combining different separation mechanisms. A typical experimental protocol includes the separation of the sample according to composition to yield fractions that are chemically homogeneous. These fractions are transferred to a size-selective separation method and analyzed with respect to molar mass. As a result of this two-dimensional (2D) separation, information on both types of molecular heterogeneity is obtained. So far, 2D chromatography has been applied mostly to polymers that are soluble inorganic solvents. There are several problems related to the use of aqueous mobile phases in polymer chromatography. These problems relate to the very polar or ionic character of the polymers and the experimental conditions, including the use of salt-containing eluents. The present paper addresses the different parameters that influence the chromatographic experiments. For a model polymer system resulting from the grafting of methacrylic acid (MAA) onto poly(ethylene glycol) (PEG), i.e., PEG-g-PMAA, it will be shown that different chromatographic techniques including SEC, LC-CC, and 2D chromatography, as well as coupled LC-CC/FT-IR can be used to analyze the molecular complexity of the copolymers.

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.