Unambiguous Tracking of Protein Phosphorylation by Fast High-Resolution FOSY NMR*.

Dysregulation of post-translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near-physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3ß) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.

Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea.

Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure-function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.

Measurement of protein backbone 13CO and 15N relaxation dispersion at high resolution.

Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics for many sites of interest in globular and intrinsically disordered proteins. We present new three-dimensional pulse sequences for measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions at backbone nitrogen and carbonyl positions. To alleviate increase in the measurement time associated with the additional spectral dimension, we use non-uniform sampling in combination with two distinct methods of spectrum reconstruction: compressed sensing and co-processing with multi-dimensional decomposition. The new methodology was validated using disordered protein CD79A from B-cell receptor and an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that, while providing much better resolution, the 3D NUS experiments give the similar accuracy and precision of the dynamic parameters to ones obtained using traditional 2D experiments. Furthermore, we show that jackknife resampling of the spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points.

A Population Shift between Sparsely Populated Folding Intermediates Determines Amyloidogenicity.

The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of nonuniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and nonamyloidogenic variants of ß2-microglobulin (ß2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the aggregation-prone ITrans state and population of a less stable, more dynamic species ablate amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding.

Tyrosine phosphorylation within the intrinsically disordered cytosolic domains of the B-cell receptor: an NMR-based structural analysis.

Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b in vitro before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.

Time-resolved multidimensional NMR with non-uniform sampling.

Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D ¹H-¹5N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.

Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.

We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution.

Backbone and ILV side chain methyl group assignments of the integral human membrane protein VDAC-1.

The voltage dependent anion channel (VDAC) forms a channel for metabolites and nutrients in the outer membrane of mitochondria, and it is also involved in apoptotic pathways. Here, we report sequence-specific NMR assignments for the isoform 1 of human VDAC reconstituted in lauryldimethylamine oxide (LDAO) detergent micelles. The assignments were deposited in the BMRB data base with accession number 16381.

Coupled decomposition of four-dimensional NOESY spectra.

Four-dimensional (4D) NOESY spectra provide unambiguous distance information at a resolution that cannot be achieved in fewer dimensions and thus increase the quality of biomolecular structure determination substantially. Since the degree of chemical shift degeneracy increases with protein size, the use of 4D NOESY spectra is particularly important for large proteins. The potential high resolution in 4D spectra cannot be achieved in a reasonable time with conventional acquisition routines that sample the Nyquist grid uniformly. It can, however, be obtained with nonuniform sampling of the data grid, but optimal processing of such data has not yet been established. Here we describe a processing method for a pair of sparsely sampled 4D NOESY spectra, a methyl-methyl and an amide-methyl NOESY, recorded on a perdeuterated protein with protonated isoleucine, leucine, and valine methyl groups. The coupled multidimensional decomposition (Co-MDD) of these two spectra together with a 2D template spectrum results in a substantial increase in sensitivity, evidenced by 50-100% additional cross peaks, when compared to alternative processing schemes. At the same time, Co-MDD allows the use of low sparse levels of 10-15% of the full data grid for NOESY spectra. For the 283-residue integral human membrane protein VDAC-1, which has a rotational correlation time of about 70 ns in detergent micelles, the two 4D Co-MDD NOESYs yielded a total of 366 NOEs, resulting in 139 unambiguous upper limit distance constraints for the structure calculation.

Solution structure of the integral human membrane protein VDAC-1 in detergent micelles.

The voltage-dependent anion channel (VDAC) mediates trafficking of small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC also interacts with antiapoptotic proteins from the Bcl-2 family, and this interaction inhibits release of apoptogenic proteins from the mitochondrion. We present the nuclear magnetic resonance (NMR) solution structure of recombinant human VDAC-1 reconstituted in detergent micelles. It forms a 19-stranded beta barrel with the first and last strand parallel. The hydrophobic outside perimeter of the barrel is covered by detergent molecules in a beltlike fashion. In the presence of cholesterol, recombinant VDAC-1 can form voltage-gated channels in phospholipid bilayers similar to those of the native protein. NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein Bcl-x(L), for reduced beta-nicotinamide adenine dinucleotide, and for cholesterol. Bcl-x(L) interacts with the VDAC barrel laterally at strands 17 and 18.

qTROSY--a novel scheme for recovery of the anti-TROSY magnetisation.

In TROSY experiments, spin state selection (S3) retains only the single HSQC sub-spectrum with minimal T2 relaxation and maximal resolution, yet at the cost of eliminating half of the available polarisation as undesired anti-TROSY component. We here introduce queued TROSY (qTROSY) as a novel scheme to partially recover and exploit this anti-TROSY polarisation in two concatenated scans. After initial orthogonal spin state separation (oS3), anti-TROSY polarisation is explicitly stored while its TROSY counterpart follows the desired coherence pathway recorded in a first scan A. The immediately appended scan B then quantitatively converts the recovered anti-TROSY polarisation into a second TROSY spectrum, skipping the time-limiting long reequilibration delay. Both concatenated qTROSY scans thus ideally exploit the full initial polarisation within almost the same measurement time. In practice, T2 relaxation losses accruing during the coupling evolution delays reduced anti-TROSY polarisation recovery below 40%, obviating sensitivity enhancement through addition of both qTROSY scans; yet, scan B retained a complete scan A spectrum with up to 75% intensity. We therefore propose to employ qTROSY asymmetrically, compacting two separate conventional into one queued TROSY-type experiment with significantly reduced measurement time, implying primarily the concatenation of different three- or higher-dimensional experiments. Both anti-TROSY polarisation recoveries and possible time savings are largest for deuterated and smaller non-deuterated proteins, extending the rentability limit of the TROSY principle towards smaller molecular weights.