The assignment of the proton and carbon spectra of the cyclic peptide cyclo(-Phe1 -Pro2 -Thr3 -Lys(Z)4 -Trp5 -Phe6 -) was accomplished by the application of multiple quantum proton-detected heteronuclear correlation spectroscopy. Since the proton spectrum shows severe overlap, the carbon chemical shifts were used to disentangle the proton resonances. The methodology described is useful even in cases where only limited quantities of materials are available. The combination of (i) a proton decoupled C,H correlation spectrum and (ii) a (not decoupled) H-relayed C,H correlation gave the assignments of all CHn groups. The non-protonated carbons, i.e. the carbonyl carbons of the peptide bond, were assigned with a C,H correlation optimized for long-range couplings, an experiment that also gave helpful information about the conformational features of the hexapeptide. The cyclic peptide contains a Phe-Pro cis-peptide bond forming a βVI-like bend and a β-turn about the amino acids Thr-Lys-Trp-Phe. Although the conventional discussion of NMR parameters indicates a strong preference for one conformation, the quantitative evaluation of NOE-derived distances in restrained MD calculations proves that the type of β-turn in the last-mentioned region is not unique. Whereas the MD calculations converge to a βII\' turn, the vicinal proton coupling constants are in better agreement with type βI. Thus a dynamic equilibrium of the backbone is proposed.

The cellulosome is a highly efficient cellulolytic complex containing cellulolytic enzymes and non-catalytic subunits, i.e. scaffoldins, which are assembled by the interactions between the dockerin modules of the enzymes and the cohesin modules of the primary scaffoldins. The cellulosome attaches to the cell surface via the S-layer homology (SLH) modules of the anchoring scaffoldins. Clostridium thermocellum DSM1313 is a thermophilic cellulosome-producing bacterium with great potential in lignocellulose bioconversion and biofuel production. The bacterium contains four anchoring scaffoldins ScaB, ScaC, ScaD and ScaF, among which ScaF is the only one that contains an additional module of unknown function (ScaF-X) between the cohesin and SLH modules. The gene of ScaF is located outside the scaffoldin gene cluster of scaA, scaB, scaC and scaD. Previous studies showed unique regulation properties and function of ScaF compared to other anchoring scaffoldins, which might be related to the additional ScaF-X module. Here we report the NMR chemical shift assignments of ScaF-X from C. thermocellum DSM1313. The well-dispersed NMR spectrum and the secondary structure prediction based on the chemical shifts of ScaF-X indicated that ScaF-X is a well-folded protein module. The chemical shift assignments provide the basis for future studies on the structure of this module and its function in cellulosomes.

Non-ribosomal peptide synthetases (NRPSs) are large multienzyme machineries. They synthesize numerous important natural products starting from amino acids. For peptide synthesis functionally specialized NRPS modules interact in a defined manner. Individual modules are either located on a single or on multiple different polypeptide chains. The \"peptide-antimicrobial-Xenorhabdus\" (PAX) peptide producing NRPS PaxS from Xenorhabdus bacteria consists of the three proteins PaxA, PaxB and PaxC. Different docking domains (DDs) located at the N-termini of PaxB and PaxC and at the C-termini of PaxA and BaxB mediate specific non-covalent interactions between them. The N-terminal docking domains precede condensation domains while the C-terminal docking domains follow thiolation domains. The binding specificity of individual DDs is important for the correct assembly of multi-protein NRPS systems. In many multi-protein NRPS systems the docking domains are sufficient to mediate the necessary interactions between individual protein chains. However, it remains unclear if this is a general feature for all types of structurally different docking domains or if the neighboring domains in some cases support the function of the docking domains. Here, we report the 1H, 13C and 15 N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (CDD) from PaxA and for its binding partner - the N-terminal docking domain (NDD) from PaxB from the Gram-negative entomopathogenic bacterium Xenorhabdus cabanillasii JM26 in their free states and for a 1:1 complex formed by the two proteins. These NMR resonance assignments will facilitate further structural and dynamic studies of this protein complex.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a 414-residue protein whose aberrant aggregation is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Intriguingly, TDP-43 has also been shown to functionally oligomerize to carry out physiological functions. TDP-43 also exists in mixed condensates or granules with other proteins (e.g. neuronal or stress granules), and its large C-terminal domain (CTD, residues 267-414) seems responsible for TDP-43 both homo- and heterotypic interactions underlying such diverse functional and pathological aggregation events. A myriad of distinct triggers may drive TDP-43 oligomerization, including interaction partners or changes in pH or salinity. In this Assignment Note, we report the complete backbone and a wealth of side chain chemical shift assignments for the CTD of TDP-43 at pH 4. The assignments presented here provide a solid starting point to study the aggregation pathway of TDP-43 at pH values below those considered physiological but relevant in pathological settings, and to contrast the aggregation behaviour under distinct conditions and in the presence of interacting partners.

Rec3 is a subdomain of the recognition (Rec) lobe within CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein Cas9 that is involved in nucleic acid binding and is critical to HNH endonuclease activation. Here, we report the backbone resonance assignments of an engineered construct of the Rec3 subdomain from Streptococcus pyogenes Cas9. We also analyze backbone chemical shift data to predict secondary structure and an overall fold that is consistent with that of Rec3 from the full-length S. pyogenes Cas9 protein.

The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a suggested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended β-sheet on one side, and another β-sheet that is distorted by loops and β-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular β-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation during biofilm formation. Here we report the near complete 15N/13C/1H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic 15N-T1, T2 and NOE data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding.

Translation initiation factor 3 (IF3) is one of the three protein factors that bind to the small ribosomal subunit and it is required for the initiation of protein biosynthesis in bacteria. IF3 contains two independent domains, N- and C-terminal domains, which are connected by a lysine-rich interdomain linker. IF3 undergoes large-scale movements and conformational changes upon binding to the 30S subunit and also during the functional regulation of initiation. However, the precise dynamic interplay of the two domains and the molecular mechanism of IF3 is not well understood. A high-resolution 3D structure of a complete IF3 in bacteria has not been solved. Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is a primary cause of nosocomial infections in humans. Here we report the NMR chemical shift assignments of IF3 from P. aeruginosa as the first step toward NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data identified nine β-strands and four α-helices arranged in the sequential order β1-β2-α1-β3-β4-α2-β5-α3-β6-α4-β7-β8-β9.

Friedreich\'s ataxia, the most prevalent hereditary ataxia, is caused by a patient\'s inability to produce a viable form of the protein frataxin. Frataxin plays an essential role in cellular iron regulation and has been shown to participate in the assembly of iron-sulfur (Fe-S) clusters under a variety of roles, including modulating persulfide production and directing Fe(II) delivery to the assembly scaffold protein. While the activity and structure of multiple eukaryotic frataxin orthologs have been characterized, the fly ortholog has numerous advantages over other orthologs with regards to protein stability, its activity towards Fe-S cluster assembly and its stability for forming stable proteins partner assemblies. Given the obvious advantages for studying the Drosophila melanogaster frataxin homolog (Dfh) over its orthologs, we have undertaken a structural characterization of apo-Dfh as the first step towards solving the solution structure of the protein alone and in complex with protein partners within the Fe-S cluster assembly pathway.

HNH is one of two endonuclease domains of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9 that perform site-specific cleavage of double-stranded DNA. We engineered a novel construct of this critical nuclease from Streptococcus pyogenes Cas9 that not only maintains the wild-type amino acid sequence and fold, but displays enhanced thermostability when compared to the full-length Cas9 enzyme. Here, we report backbone and side chain assignments of the HNH nuclease as a foundational step toward the characterization of protein dynamics and allostery in CRISPR-Cas9.

Retinoblastoma-binding protein 1 (RBBP1), also known as AT-rich interaction domain 4A (ARID4A), is a tumour suppressor involved in the regulation of the epigenetic programming in leukemia and Prader-Willi/Angelman syndromes. The ARID domain of RBBP1 binds to DNA non-specifically and has gene suppression activity. However, no structural data has been obtained for the human RBBP1 ARID domain so far. Here we report the near-complete 1H, 13C, 15N backbone and side-chain NMR assignment of a 27 kDa tandem PWWP-ARID domain construct that spans residues 171-414 with the removal of a short disordered region between the two domains. The predicted secondary structure based on the assigned chemical shifts is consistent with the structures of the isolated PWWP domain of human RBBP1 previously solved and the homologous ARID domains of other proteins.