A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein,an intrinsically disordered protein

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H^N–H^α and H^N–H^N NOEs, values for ³J_HNHα, ¹J_HαCα, ²J_CαN, and ¹J_CαN, as well as chemical shifts of ¹⁵N, ¹³C^α, and ¹³C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PP_II region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the α_R region (10–20%) is found. Analysis of ¹H–¹H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.     

1H, 15N and 13C backbone resonance assignments of the archetypal serpin α1-antitrypsin

Alpha(1)-antitrypsin is a 45-kDa (394-residue) serine protease inhibitor synthesized by hepatocytes, which is released into the circulatory system and protects the lung from the actions of neutrophil elastase via a conformational transition within a dynamic inhibitory mechanism. Relatively common point mutations subvert this transition, causing polymerisation of α(1)-antitrypsin and deficiency of the circulating protein, predisposing carriers to severe lung and liver disease. We have assigned the backbone resonances of α(1)-antitrypsin using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for a detailed solution state characterization of the structural properties of this highly dynamic protein via NMR methods.     

[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N–1H, aromatic 13C–1H and side-chain 15N–1H2 correlations in large proteins

This paper describes a [¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide ¹⁵N–¹H groups, side chain ¹⁵N–¹H₂ groups and aromatic ¹³C–¹H groups in otherwise highly deuterated proteins. The ¹⁵N–¹H and ¹³C–¹H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the ¹H dimension. The side-chain ¹⁵N–¹H₂ groups, which are suppressed in conventional [¹⁵N,¹H)-TROSY, are observed with high sensitivity in the ¹⁵N–¹H subspectrum. [¹⁵N,¹H]/[¹³C,¹H]-TROSY was used as the heteronuclear correlation block in a 3D [¹H,¹H]-NOESY-[¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain ¹⁵N–¹H₂ groups.     

Segmental conformational disorder and dynamics in the intrinsically disordered protein α-synuclein and its chain length dependence

Conformational ensembles of fully disordered natural polypeptides represent the starting point of protein refolding initiated by transfer to folding conditions. Thus, understanding the transient properties and dimensions of such peptides under folding conditions is a necessary step in the understanding of their subsequent folding behavior. Such ensembles can also undergo alternative folding and form amyloid structures, which are involved in many neurological degenerative diseases. Here, we performed a structural study of this initial state using time-resolved fluorescence resonance energy transfer analysis of a series of eight partially overlapping double-labeled chain segments of the N-terminal and NAC domains of the α-synuclein molecule. The distributions of end-to-end distance and segmental intramolecular diffusion coefficients were simultaneously determined for eight labeled chain segments. We used the coefficient of variation, C_v, as a measure of the conformational heterogeneity (i.e., structural disorder). With the exception of two segments, the C_vs were characteristic of a fully disordered state of the chain. Subtle deviations from this behavior at the segment labeled in the NAC domain and the segment at the N termini reflected subtle conformational bias that might be related to the initiation of transition to amyloid aggregates. The chain length dependence of the mean segmental end-to-end distance followed a power law as predicted by Flory, but the dependence was steeper than previously predicted, probably due to the contribution of the excluded volume effect, which is more dominant for shorter-chain segments. The observed intramolecular diffusion coefficients (< 10 to ∼ 25 ?²/ns) are only an order of magnitude lower than the common diffusion coefficients of low molecular weight probes. This diffusion coefficient increased with chain length, probably due to the cumulative contributions of minor bond rotations along the chain. These results gave us a reference both for characteristics of a natural unfolded polypeptide at the moment of initiation of folding and for detection of possible initiation sites of the amyloid transition.     

1H, 13C, and 15N backbone and side-chain chemical shift assignments for the 29 kDa human galectin-1 protein dimer

Galectin-1 is an important regulator of leukocyte function and tumor angiogenesis. Recently, this lectin has been identified as a molecular target for the potent angiogenesis inhibitor anginex. Here, we report ¹H, ¹³C, and ¹⁵N chemical shift assignments for human galectin-1 as determined by using heteronuclear triple resonance NMR spectroscopy.     

Accurate measurements of the effects of deuteration at backbone amide positions on the chemical shifts of 15N, 13Cα, 13Cβ, 13CO and 1Hα nuclei in proteins

An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins (¹⁵N, ¹³C_α, ¹³CO, ¹H_α) and ¹³C_β nuclei arising from ¹H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond ¹³C_α isotope shifts, ²ΔC_α(N_iD) and ³ΔC_α(N_i+1D), and three-bond ¹³C_β isotope shifts, ³ΔC_β(N_iD), are useful reporters of the local geometry of the protein backbone.     

1H, 13C and 15N assignments of a camelid nanobody directed against human α-synuclein

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens (Kd ~ nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human α-synuclein (aS), whose aberrant self-association is implicated in Parkinson’s disease.     

The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.     

1H, 13C, and 15N resonance assignments for human regenerating family Iα protein

Human regenerating (Reg) genes belong to the C-type lectin superfamily and express secretory proteins in various tissues. Reg Iα, also named lithostathine, has multiple roles in numerous biological events such as cytokines, anti-apoptotic factors and the calcium carbonate crystals inhibitor. Under physiological pH, Reg Iα becomes largely insoluble after a self-proteolysis process, and the N-terminally truncated form readily polymerizes into fibrils, which leads to neurodegenerative diseases. Reg Iα may form protofibril via lateral hydrophobic interactions with a native-like conformation. The structural basis from the native to fibril form, as well as the carbohydrate binding sites on Reg Iα, remain unknown. Here we present the NMR backbone and side-chain assignments of Reg Iα for use in further NMR investigations.     

1H, 13C and 15N backbone and side chain resonance assignments of human interleukin 1α

As part of our NMR structure determination of the human Interleukin-1α, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C and ¹⁵N nuclei.     

1H, 13C and 15N resonance assignments of α-domain for Bacillus subtilis Lon protease

The small α-domain of Lon protease is thought to carry the substrate-recognition, nucleotide-binding, and DNA-binding sites. Here we report the complete resonance assignment of the α-domain for Bacillus subtilis Lon protease (Bs-Lon α-domain).     

Assignment of protein backbone resonances using connectivity, torsion angles and 13Cα chemical shifts

A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just (13)C(alpha) chemical shifts (delta((13)C(alpha))) and data restricting the phi and psi backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in delta((13)C(alpha)), phi, and psi space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and (13)C(alpha) chemical shift data are available.     

Differential deuterium isotope shifts and one-bond 1H−13C scalar couplings in the conformational analysis of protein glycine residues

Summary The one-bond deuterium isotope shift effect for glycine C resonances exhibits a conformational dependence comparable to that of the corresponding ¹J_HC scalar coupling in both magnitude (11 Hz at 14.1 T) and dihedral angle dependence. The similarity in the conformational dependence of the ¹J_HC and deuterium isotope shift values suggests a common physical basis. Given the known distribution of (,) main-chain dihedral angles for glycine residues, the deuterium isotope shifts and the ¹J_HC scalar couplings can determine conformations in the left-and right-handed helical-to-bridge regions of the (,) plane to an accuracy of approximately 13°. In the absence of stereochemical assignments, the differential deuterium isotope shifts and the ¹J_HC scalar couplings can be combined with limited independent structural information (e.g., the sign of ) to determine the chirality of the deuterium substitution.     

1H, 13C and 15N backbone resonance assignments for the BS3 class A β-lactamase from Bacillus licheniformis

Class A β-lactamases (260–280 amino acids; M _r  ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-α domain formed by five to eight helices and an α/β domain consisting of a five-stranded antiparallel β-sheet covered by three to four α-helices. The α domain (~150 residues) is made up of the central part of the polypeptide chain whereas the α/β domain (111–135 residues) is constituted by the N- and C-termini of the protein. Our goal is to determine in which order the different secondary structure elements are formed during the folding of BS3. With this aim, we will use pulse-labelling hydrogen/deuterium exchange experiments, in combination with 2D-NMR measurements, to monitor the time-course of formation and stabilization of secondary structure elements. Here we report the backbone resonance assignments as the requirement for further hydrogen/deuterium exchange studies.     

Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R₁, R_1ρ, R₂ and η_xy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.     

Backbone and Ile-δ1, Leu, Val Methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein

Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone ¹H^N, ¹⁵N, ¹³C′, and ¹³C^α, as well as side chain ¹³C^β, methyl (Ile-δ1, Leu, Val), amide (Asn, Gln), and indole N–H (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.     

MQ-HCN-based pulse sequences for the measurement of 13C1′-1H1′, 13C1′-15N, 1H1′-15N, 13C1′-13C2′, 1H1′-13C2′, 13C6/8-1H6/8, 13C6/8-15N, 1H6/8-15N, 13C6-13C5, 1H6-13C5 dipolar couplings in 13C, 15N-labeled DNA (and RNA)

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.