<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of human osteopontin

Osteopontin (OPN) is a 33.7 kDa intrinsically disordered protein and a member of the SIBLING family of proteins. OPN is bearing a signal peptide for secretion into the extracellular space, where it exerts its main physiological function, the control of calcium biomineralization. It is often involved in tumorigenic processes influencing proliferation, migration and survival, as well as the adhesive properties of cancer cells via CD44 and integrin signaling pathways. Here we report the nearly complete NMR chemical shift assignment of recombinant human osteopontin.     

Sequence-specific <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of the autophagy-related protein LC3C

Autophagy is a versatile catabolic pathway for lysosomal degradation of cytoplasmic material. While the phenomenological and molecular characteristics of autophagic non-selective (bulk) decomposition have been investigated for decades, the focus of interest is increasingly shifting towards the selective mechanisms of autophagy. Both, selective as well as bulk autophagy critically depend on ubiquitin-like modifiers belonging to the Atg8 (autophagy-related 8) protein family. During evolution, Atg8 has diversified into eight different human genes. While all human homologues participate in the formation of autophagosomal membrane compartments, microtubule-associated protein light chain 3C (LC3C) additionally plays a unique role in selective autophagic clearance of intracellular pathogens (xenophagy), which relies on specific protein–protein recognition events mediated by conserved motifs. The sequence-specific ¹H, ¹⁵N, and ¹³C resonance assignments presented here form the stepping stone to investigate the high-resolution structure and dynamics of LC3C and to delineate LC3C’s complex network of molecular interactions with the autophagic machinery by NMR spectroscopy.     

Backbone <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of the Tom1 VHS domain

Efficient trafficking of ubiquitinated receptors (cargo) to endosomes requires the recruitment of adaptor proteins that exhibit ubiquitin-binding domains for recognition and transport. Tom1 is an adaptor protein that not only associates with ubiquitinated cargo but also represents a phosphoinositide effector during specific bacterial infections. This phosphoinositide-binding property is associated with its N-terminal Vps27, Hrs, STAM (VHS) domain. Despite its biological relevance, there are no resonance assignments of Tom1 VHS available that can fully characterize its molecular interactions. Here, we report the nearly complete ¹H, ¹⁵N, and ¹³C backbone resonance assignments of the VHS domain of human Tom1.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of human guanylate kinase

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain ¹H, ¹³C, and ¹⁵N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

NMR <Superscript>1</Superscript>H,<Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and <Superscript>13</Superscript>C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here ¹H^{N, 15}N, and ¹³C resonance assignments for the 19.3 kDa (aa 1–169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS^G12C-GDP), using heteronuclear, multidimensional NMR spectroscopy. Backbone ¹H–¹⁵N correlations have been assigned for all non-proline residues, except for the first methionine residue.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a C-terminal domain of human CHD1

Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C sequence specific backbone assignment of the vanadate inhibited hematopoietic tyrosine phosphatase

The sequence-specific backbone assignment of hematopoietic protein tyrosine phosphatase (HePTP; PTPN7) in presence of vanadate has been determined, based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. These assignments facilitate further studies of HePTP in the presence of inhibitors to target leukemia and provide further insights into the function of protein tyrosine phosphatases.     

<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments of light organ-associated fatty acid-binding protein of Taiwanese fireflies

Fatty acid-binding proteins (FABPs) are a family of proteins that modulate the transfer of various fatty acids in the cytosol and constitute a significant portion in many energy-consuming cells. The ligand binding properties and specific functions of a particular type of FABP seem to be diverse and depend on the respective binding cavity as well as the cell type from which this protein is derived. Previously, a novel FABP (lcFABP; lc: Luciola cerata) was identified in the light organ of Taiwanese fireflies. The lcFABP was proved to possess fatty acids binding capabilities, especially for fatty acids of length C₁₄–C₁₈. However, the structural details are unknown, and the structure–function relationship has remained to be further investigated. In this study, we finished the ¹H, ¹⁵N and ¹³C chemical shift assignments of ¹⁵N/¹³C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, C_α, H_α, C and side chain C_β assignments. These results can provide the basis for further structural exploration of lcFABP.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C assignment of the amyloidogenic protein medin using fast-pulsing NMR techniques

Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments of FAS1-IV domain of human periostin,a component of extracellular matrix proteins

Periostin, an extracellular matrix protein, is secreted by fibroblasts and is overexpressed in various types of cancers. The four internal repeat fasciclin 1 (FAS1) domains of human periostin play crucial roles in promoting tumor metastasis and progression via interaction with cell surface integrins. Among four FAS1 domains of human periostin, the fourth FAS1 domain (FAS1-IV) was prepared for NMR study, since only FAS1-IV was highly soluble, and showed a well-dispersed 2D ¹H-¹⁵N HSQC spectrum. Here, we report nearly complete backbone and side chain resonance assignments and a secondary structural analysis of the FAS1-IV domain as first steps toward the structure determination of FAS1-IV of human periostin.