Alessandro Piai

Speeding up sequence specific assignment of IDPs

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct 13C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

The crowd you're in with: Effects of different types of crowding agents on protein aggregation

The intracellular environment contains high concentrations of macromolecules occupying up to 30% of the total cellular volume. Presence of these macromolecules decreases the effective volume available for the proteins in the cell and thus increases the effective protein concentrations and stabilizes the compact protein conformations. Macromolecular crowding created by various macromolecules such as proteins, nucleic acids, and carbohydrates has been shown to have a significant effect on a variety of cellular processes including protein aggregation. Most studies of macromolecular crowding have used neutral, flexible polysaccharides that function primarily via excluded volume effect as model crowding agents. Here we have examined the effects of more rigid polysaccharides on protein structure and aggregation. Our results indicate that rigid and flexible polysaccharides influence protein aggregation via different mechanisms and suggest that, in addition to excluded volume effect, changes in solution viscosity and non-specific protein-polymer interactions influence the structure and dynamics of proteins in crowded environments.

High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins

We present three novel exclusively heteronuclear 5D 13C direct-detected NMR experiments, namely (HN-flipN)CONCACON, (HCA)CONCACON and (H)CACON(CA)CON, designed for easy sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The experiments proposed have been optimized to overcome the drawbacks which may dramatically complicate the characterization of IDPs by NMR, namely the small dispersion of chemical shifts and the fast exchange of the amide protons with the solvent. A fast and reliable automatic assignment of α-synuclein chemical shifts was obtained with the Tool for SMFT-based Assignment of Resonances (TSAR) program based on the information provided by these experiments.

“CON-CON” assignment strategy for highly flexible intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel 1H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on 13C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.

NMR Methods for the Study of Instrinsically Disordered Proteins Structure, Dynamics, and Interactions: General Overview and Practical Guidelines

Thanks to recent improvements in NMR instrumentation, pulse sequence design, and sample preparation, a panoply of new NMR tools has become available for atomic resolution characterization of intrinsically disordered proteins (IDPs) that are optimized for the particular chemical and spectroscopic properties of these molecules. A wide range of NMR observables can now be measured on increasingly complex IDPs that report on their structural and dynamic properties in isolation, as part of a larger complex, or even inside an entire living cell. Herein we present basic NMR concepts, as well as optimised tools available for the study of IDPs in solution. In particular, the following sections are discussed hereafter: a short introduction to NMR spectroscopy and instrumentation (Sect. 3.1), the effect of order and disorder on NMR observables (Sect. 3.2), particular challenges and bottlenecks for NMR studies of IDPs (Sect. 3.3), 2D HN and CON NMR experiments: the fingerprint of an IDP (Sect. 3.4), tools for overcoming major bottlenecks of IDP NMR studies (Sect. 3.5), 13C detected experiments (Sect. 3.6), from 2D to 3D: from simple snapshots to site-resolved characterization of IDPs (Sect. 3.7), sequential NMR assignment: 3D experiments (Sect. 3.8), high-dimensional NMR experiments (nD, with n>3) (Sect. 3.9) and conclusions and perspectives (Sect. 3.10).

Sequence Context Influences the Structure and Aggregation Behavior of a PolyQ Tract

Expansions of polyglutamine (polyQ) tracts in nine different proteins cause a family of neurodegenerative disorders called polyQ diseases. Because polyQ tracts are potential therapeutic targets for these pathologies there is great interest in characterizing the conformations that they adopt and in understanding how their aggregation behavior is influenced by the sequences flanking them. We used solution NMR to study at single-residue resolution a 156-residue proteolytic fragment of the androgen receptor that contains a polyQ tract associated with the disease spinobulbar muscular atrophy, also known as Kennedy disease. Our findings indicate that a Leu-rich region preceding the polyQ tract causes it to become α-helical and appears to protect the protein against aggregation, which represents a new, to our knowledge, mechanism by which sequence context can minimize the deleterious properties of these repetitive regions. Our results have implications for drug discovery for polyQ diseases because they suggest that the residues flanking these repetitive sequences may represent viable therapeutic targets.

Just a Flexible Linker? The Structural and Dynamic Properties of CBP-ID4 Revealed by NMR Spectroscopy

Here, we present a structural and dynamic description of CBP-ID4 at atomic resolution. ID4 is the fourth intrinsically disordered linker of CREB-binding protein (CBP). In spite of the largely disordered nature of CBP-ID4, NMR chemical shifts and relaxation measurements show a significant degree of α-helix sampling in the protein regions encompassing residues 2-25 and 101-128 (1852-1875 and 1951-1978 in full-length CBP). Proline residues are uniformly distributed along the polypeptide, except for the two α-helical regions, indicating that they play an active role in modulating the structural features of this CBP fragment. The two helical regions are lacking known functional motifs, suggesting that they represent thus-far uncharacterized functional modules of CBP. This work provides insights into the functions of this protein linker that may exploit its plasticity to modulate the relative orientations of neighboring folded domains of CBP and fine-tune its interactions with a multitude of partners.

Chapter Six - Recent Advances in Solution NMR Studies: 13C Direct Detection for Biomolecular NMR Applications

Abstract Carbon-13 direct detection NMR provides a complementary tool for biomolecular applications, thanks to the development of a large variety of experimental variants and to recent improvements in instrumental sensitivity. It can be used as a general tool for any protein study and it can provide unique information very relevant for the characterization of a variety of different systems such as paramagnetic proteins and large multimeric protein assemblies, as well as intrinsically disordered proteins. The different properties of 13 C respect to 1 H, which provide the rational for the experiments developed, are discussed. The technical aspects that needed to be solved, as well as the many experimental variants developed to address different cases, are presented. Application areas where these experiment result particularly useful are also described.

Optimal Bicelle Size q for Solution NMR Studies of the Protein Transmembrane Partition.

Structural characterization of transmembrane proteins in isotropic bicelles has become an increasingly popular application of solution NMR spectroscopy, as the fast-tumbling bicelles are membrane-like, yet can often yield spectral quality comparable to those of detergent micelles. While larger bicelles are closer to the true lipid bilayer, it remains unclear how large the bicelles need to be to allow accurate assessment of the protein transmembrane partition in the lipid bilayer. Here, we address the above question from the perspective of the protein residing in the bicelles, through systematic measurement of the protein chemical shift and transmembrane partition at different lipid/detergent ratios (q), ranging from 0.3 to 0.7, using the transmembrane domain of the human Fas receptor as model system. We found that the lipid environment of the bicelles, as reflected by the protein chemical shift, begins to be perturbed when q is reduced to below 0.6. We also implemented a solvent paramagnetic relaxation enhancement (PRE) approach for bicelles to show that the protein transmembrane partition in bicelles with q=0.5 and 0.7 are very similar, but at q=0.3 the solvent PRE profile is significantly different. Our data indicate that q values between 0.5 and 0.6 are a good compromise between high resolution NMR and closeness to the membrane environment, and allow accurate characterization of the protein position in the lipid bilayer.

Amino acid recognition for automatic resonance assignment of intrinsically disordered proteins

Resonance assignment is a prerequisite for almost any NMR-based study of proteins. It can be very challenging in some cases, however, due to the nature of the protein under investigation. This is the case with intrinsically disordered proteins, for example, whose NMR spectra suffer from low chemical shifts dispersion and generally low resolution. For these systems, sequence specific assignment is highly time-consuming, so the prospect of using automatic strategies for their assignment is very attractive. In this article we present a new version of the automatic assignment program TSAR dedicated to intrinsically disordered proteins. In particular, we demonstrate how the automatic procedure can be improved by incorporating methods for amino acid recognition and information on chemical shifts in selected amino acids. The approach was tested in silico on 16 disordered proteins and experimentally on α-synuclein, with remarkably good results.