Biomolecular NMR Assignments最新文献

Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR’s research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the ¹H,¹⁵N,¹³C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of β-sheets corresponding to around 40% of the B2-3 backbone. The Cβ chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy.

Amyloid fibrils from Alzheimer’s amyloid-beta peptides (Aβ) are found to be polymorphic. So far, 14 Aβ40 fibril structures have been determined. The mechanism of why one particular protein sequence adopts so many different three-dimensional structures is yet not understood. In this work, we describe the assignment of the NMR chemical shifts of two Alzheimer’s disease fibril polymorphs, P1 and P2, which are formed by the amyloid-beta peptide Aβ40. The assignment is based on ¹³C-detected 3D NCACX and NCOCX experiments MAS solid-state NMR experiments. The fibril samples are prepared using an extensive seeding protocol in the absence and presence of the small heat shock protein αB-crystallin. In addition to manual assignments, we obtain chemical shift assignments using the automation software ARTINA. We present an analysis of the secondary chemical shifts and a discussion on the differences between the manual and automated assignment strategies.

Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein’s N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the ¹³C,¹⁵N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176–181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies.

Synucleinopathies are neurodegenerative diseases characterized by the accumulation of α-synuclein protein aggregates in the neurons and glial cells. Both ex vivo and in vitro α-synuclein fibrils tend to show polymorphism. Polymorphism results in structure variations among fibrils originating from a single polypeptide/protein. The polymorphs usually have different biophysical, biochemical and pathogenic properties. The various pathologies of a single disease might be associated with distinct polymorphs. Similarly, in the case of different synucleinopathies, each condition might be associated with a different polymorph. Fibril formation is a nucleation-dependent process involving the formation of transient and heterogeneous intermediates from monomers. Polymorphs are believed to arise from heterogeneous oligomer populations because of distinct selection mechanisms in different conditions. To test this hypothesis, we isolated and incubated different intermediates during in vitro fibrillization of α-synuclein to form different polymorphs. Here, we report ¹³C and ¹⁵N chemical shifts and the secondary structure of fibrils prepared from the helical intermediate using solid-state nuclear magnetic spectroscopy.

The α-synuclein (α-syn) amyloid fibrils are involved in various neurogenerative diseases. Solid-state NMR (ssNMR) has been showed as a powerful tool to study α-syn aggregates. Here, we report the ¹H, ¹³C and ¹⁵N back-bone chemical shifts of a new α-syn polymorph obtained using proton-detected ssNMR spectroscopy under fast (95 kHz) magic-angle spinning conditions. The manual chemical shift assignments were cross-validated using FLYA algorithm. The secondary structural elements of α-syn fibrils were calculated using ¹³C chemical shift differences and TALOS software.

Adalimumab is a therapeutic monoclonal antibody developed to target human TNF an important mediator of immune-mediated inflammatory diseases such as rheumatoid arthritis, amongst others. The 48 kDa Fab fragment of adalimumab was produced in Escherichia coli using a single chain approach to allow complete isotopic incorporation of deuterium, carbon-13 and nitrogen-15 along with the protonated isoleucine-d, valine and leucine methyl groups. Here we report the near complete resonance assignment of the polypeptide backbone and the methyl groups of isoleucine, leucine and valine residues.

Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa, and recently isolated cases worldwide, there are no current therapeutics or vaccines. As such, LASV poses a significant global public health threat. One of the key steps in LASV infection is delivering its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Previously, we showed that the TM undergoes pH-dependent structural changes that result in a helical extension. Here, we provide the ¹H, ¹⁵N, and ¹³C assignment of the LASV TM backbone in the prefusion and postfusion states. We also provide the ¹H, ¹⁵N, and ¹³C assignment of two mutants, G429P and D432P, which prevent this helical extension. These results will help understand the role the TM plays in membrane fusion and can lead to the design of therapeutics against LASV infection.

Signalosomes are high-order protein machineries involved in complex mechanisms controlling regulated immune defense and cell death execution. The immune response is initiated by the recognition of exogeneous or endogenous signals, triggering the signalosome assembly process. The final step of signalosome fate often involves membrane-targeting and activation of pore-forming execution domains, leading to membrane disruption and ultimately cell death. Such cell death-inducing domains have been thoroughly characterized in plants, mammals and fungi, notably for the fungal cell death execution protein domain HeLo. However, little is known on the mechanisms of signalosome-based immune response in bacteria, and the conformation of cell death executors in bacterial signalosomes is still poorly characterized. We recently uncovered the existence of NLR signalosomes in various multicellular bacteria and used genome mining approaches to identify putative cell death executors in Streptomyces olivochromogenes. These proteins contain a C-terminal amyloid domain involved in signal transmission and a N-terminal domain, termed BELL for Bacteria analogous to fungal HeLL (HeLo-like), presumably responsible for membrane-targeting, pore-forming and cell death execution. In the present study, we report the high yield expression of S. olivochromogenes BELL2 and its characterization by solution NMR spectroscopy. BELL is folded in solution and we report backbone and sidechain assignments. We identified five α-helical secondary structure elements and a folded core much smaller than its fungal homolog HeLo. This study constitutes the first step toward the NMR investigation of the full-length protein assembly and its membrane targeting.

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA₂) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain.

A glycoside hydrolase family 45 (GH45) enzyme from the white-rot basidiomycete fungus Phanerochaete chrysosporium (PcCel45A) was expressed in Pichia pastoris with ¹³C and ¹⁵N labelling. A nearly complete assignment of ¹H, ¹³C and ¹⁵N backbone resonances was obtained, as well as the secondary structure prediction based on the assigned chemical shifts using the TALOS-N software. The predicted secondary structure was almost identical to previously published crystal structures of the same enzyme, except for differences in the termini of the sequence. This is the first NMR study using an isotopically labelled GH45 enzyme.