Proline-directed random-coil chemical shift values as a tool for the NMR assignment of the tau phosphorylation sites

NMR spectroscopy of the full-length neuronal Tau protein has proved to be difficult due to the length of the protein and the unfavorable amino acid composition. We show that the random-coil chemical shift values and their dependence on the presence of a proline residue in the (i+1) position can successfully be exploited to assign all proline-directed phosphorylation sites. This is a first step toward the study of the phosphorylation of Tau by NMR spectroscopy.     

NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase

Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.     

Regions of tau implicated in the paired helical fragment core as defined by NMR

We have studied the mature Alzheimer-like fibers of tau by fluorescence and NMR spectroscopy. Assembly of the protein into paired helical filaments after incubation with heparin at 37 degrees C was verified by electron microscopy and size-exclusion chromatography. NMR spectroscopy on these mature fibers revealed different regions of residual mobility for tau: the N-terminal domain was found to maintain solution-like dynamics and was followed by a large domain of decreasing mobility; finally the core region was distinguished by a solid-like character. Heteronuclear-NOE data indicate that the decreasing mobility is due to both a slowing down of the rapid nanosecond movements and the introduction of slower movements that lead to exchange broadening. Fluorescence spectroscopy confirmed the presence of this rigid core, and some degree of protection from hydrogen exchange for those residues was observed. Hence, our data give a more precise picture of the dynamics of tau when it is integrated into mature filaments and should provide further understanding of the molecular processes that govern aggregation.     

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution ¹⁹F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.     

In-cell solid-state NMR as a tool to study proteins in large complexes

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.     

Impact of the C‐terminal Disulfide Bond on the Folding and Stability of Onconase

The two homologous proteins ribonuclease A and onconase fold through conserved initial contacts but differ significantly in their thermodynamic stability. A disulfide bond is located in the folding initiation site of onconase (the C‐terminal part of the protein molecule) that is missing in ribonuclease A, whereas the other three disulfide bonds of onconase are conserved in ribonuclease A. Consequently, the deletion of this C‐terminal disulfide bond (C87–C104) allows the impact of the contacts in this region on the folding of onconase to be studied. We found the C87A/C104A‐onconase variant to be less active and less stable than the wild‐type protein, whereas the tertiary structure, which was determined by both X‐ray crystallography and NMR spectroscopy, was only marginally affected. The folding kinetics of the variant, however, were found to be changed considerably in comparison to wild‐type onconase. Proton exchange experiments in combination with two‐dimensional NMR spectroscopy revealed differences in the native‐state dynamics of the two proteins in the folding initiation site, which are held responsible for the changed folding mechanism. Likewise, the molecular dynamics simulation of the unfolding reaction indicated disparities for both proteins. Our results show that the high stability of onconase is based on the efficient stabilization of the folding initiation site by the C‐terminal disulfide bond. The formation of the on‐pathway intermediate, which is detectable during the folding of the wild‐type protein and promotes the fast and efficient refolding reaction, requires the presence of this covalent bond.     

A Substantial Structural Conversion of the Native Monomer Leads to in-Register Parallel Amyloid Fibril Formation in Light-Chain Amyloidosis

Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show β-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially ¹⁵N:¹³C-labeled samples, that the β-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.     

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

Abstract: 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.     

Past and Future Solid-State NMR Spectroscopy Studies at the Convergence Point between Biology and Materials Research

Many cellular events involve attachment of proteins to the surfaces of rigid or semi-rigid solid materials, such as the inorganic materials in the extracellular matrix of hard tissue, and the macromolecular scaffolds made of actin and tubulin filaments in the cytoskeleton. Understanding these processes on a fundamental level will have far-reaching repercussions for the design of biomaterials, biomedical research, and biomineralization. Numerous studies have reported structural changes experienced by proteins as they adhere to surfaces, yet there are only a few examples in which detailed views of protein conformation and alignment on surfaces were measured. Modern multidimensional solid-state NMR spectroscopy is timely situated to unveil molecular details of these processes and shed light on many fundamental questions related to recognition of surfaces by biomolecules. Targeting these questions is currently at the focal point of many research fields and can lead to insights and breakthroughs in biotechnology and in biomimetic material design.     

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy

Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low‐temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin‐T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long‐range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.     

A fluorogenic peptide containing the processing site of human SARS corona virus S-protein: kinetic evaluation and NMR structure elucidation

Human severe acute respiratory syndrome coronavirus (hSARS-CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host-virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS-CoV spike protein (Abz-(755)Glu-Gln-Asp-Arg-Asn-Thr-Arg-Glu-Val-Phe-Ala-Gln(766)-Tyx-NH(2)) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC-inhibitor, alpha1-PDX, in a dose-dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. (1)H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C-terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R(761) E has been proposed.     

Solution NMR studies of an intrinsically unstructured protein within a dilute, 75 kDa eukaryotic protein assembly; probing the practical limits for efficiently assigning polypeptide backbone resonances

This paper describes an efficient NMR strategy for assigning the backbone resonances of an intrinsically unstructured protein (IUP), p21-KID, bound to its biological target, Cdk2/cyclin A. In order to overcome the challenges associated with the high molecular weight (75 kDa) and low solubility of the ternary complex (0.2 mM), we used perdeuteration, TROSY, and high-sensitivity cryogenic NMR probes at high magnetic-field strengths (i.e. 16.4, 18.8 and 21.1 Tesla). p21-KID was also prepared by using specific amino acid isotope labels. Most importantly, we studied binary, subcomplexes that allowed resonance assignments to be made in stages. We show that subdomains of p21-KID folded within binary complexes into the same conformations as observed in the ternary, Cdk2/cyclin A complex. This is a general feature of IUPs, which often adopt highly extended conformations when bound to other proteins. This strategy is suitable for studies of IUPs within considerably larger biomolecular assemblies as long as the IUP can be uniformly and selectively isotope labeled.