Toho-1 ß-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam.

Backbone chemical shift assignments for the Toho-1 ß-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly 2H,13C,15N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-ß-lactam ß-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of 15N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR.

Backbone assignments for the isolated α-subunit of Salmonella typhimurium tryptophan synthase (TS) are reported based on triple resonance solution-state NMR experiments on a uniformly 2H,13C,15N-labeled sample. From the backbone chemical shifts, secondary structure and random coil index order parameters (RCI-S2) are predicted. Titration with the 3-indole-D-glycerol 3'-phosphate analog, N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), leads to chemical shift perturbations indicative of conformational changes from which an estimate of the dissociation constant is obtained. Comparisons of the backbone chemical-shifts, RCI-S2 values, and site-specific relaxation times with and without F9 reveal allosteric changes including modulation in secondary structures and loop rigidity induced upon ligand binding. A comparison is made to the X-ray crystal structure of the α-subunit in the full TS αßßα bi-enzyme complex and to two new X-ray crystal structures of the isolated TS α-subunit reported in this work.

Backbone and side chain 1H, 15N and 13C chemical shift assignments of the molten globule state of L94G mutant of horse cytochrome-c.

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25 °C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25 °C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments.

Backbone and side chain resonance assignments of the C-terminal domain of human TGIF1.

TGIF1 is an essential regulator of cell differentiation in various biological processes, and is associated with holoprosencephaly and many cancers. The C-terminal domain of TGIF1 that was originally defined as repressive domain 2 can interact with a variety of proteins, such as transcription factor Smad2 and co-repressor Sin3A, to mediate the regulative roles of TGIF1 in diverse cell signaling pathways. However, the recognition mechanism of TGIF1 C-terminal domain for different interacting proteins remains unknown. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of TGIF1 C-terminal domain (residues 256-375), laying a foundation for further research on the structure-function relationship of TGIF1.

Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery.

Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain 1H, 13C and 15N NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.

1H, 15N and 13C resonance assignments of the C-terminal domain of the P protein of the Nishigahara strain of rabies virus.

The C-terminal domain of the P protein of rabies virus is a multifunctional domain that interacts with both viral and host cell proteins. Here we report the 1H, 13C and 15N chemical shift assignments of this domain from P protein of the Nishigahara strain of rabies virus, a pathogenic laboratory strain well established for studies of virulence functions of rabies virus proteins, including P protein. The data and secondary structure analysis are in good agreement with the reported predominantly helical structure of the same domain from the CVS strain of rabies solved by crystallography. These assignments will enable future solution studies of the interactions of the P protein with viral and host proteins, and the effects of post-translational modifications.

Backbone and side-chain chemical shift assignments of MarH, a critical intermediary epimerase for biosynthesis of Maremycins in Streptomyces.

MarH is an essential epimerase that catalyzes the isomerization of 3R-ß-methyl-indolepyruvate (ß-MeInPy) to 3S-ß-MeInPy, which is the important precursor for biosynthesis of Maremycins. Biophysical study of the structure of MarH would be informative for better understanding of its catalytic mechanism and feasible application of the enzyme in isomerization reaction. Here, we report the backbone and side-chain NMR chemical shift assignments of MarH, which lays a foundation for further structural and mechanical study of the enzyme.

Chemical shift assignments of RHE_RS02845, a NTF2-like domain-containing protein from Rhizobium etli.

The nuclear transport factor 2 (NTF2) like superfamily includes members of the NTF2 family, delta-5-3-ketosteroid isomerases, and the beta subunit of ring hydroxygenases. This family plays important roles in both eukaryotic and prokaryotic cells, and is taken as a classic example of divergent evolution because proteins in this family exhibit diverse biological functions, although share common structural features. We cloned the gene RHE_RS02845 encoding a predicted NTF2-like domain-containing protein in Rhizobium etli, and prepared U-13C/15N-labeled protein samples for its three-dimensional NMR structural determination. Here, chemical shift assignments for both backbone and side-chain atoms are reported, which is prerequisite for further structural calculation and functional research using NMR spectroscopy.

Chemical shift assignments of CHU_1110: an AHSA1-like protein from Cytophaga hutchinsonii.

AHSA1 protein family is one of the four largest families in the Bet v1-like protein superfamily. The functions and structures of proteins in AHSA1 family are still largely unknown. CHU_1110 with 167 amino acids and a molecular weight of 19.2 kDa is a member of the AHSA1 family from Cytophaga hutchinsonii, a soil bacterium known for its ability to digest crystalline cellulose. Here we report the complete 1H, 13C and 15N chemical shift assignments of CHU_1110. The secondary structural elements of CGL2373 are consistent with the canonical AHSA1 structure. However the sequence identity of CHU_1110 with other members of AHSA1 family with functional and structural reports, such as RHE_CH02687 from Rhizobium etli, Aha1 from Homo sapiens and Yndb from Bacillus subtilis, are very low, which may suggest a different function of CHU_1110. Our chemical shift assignments of CHU_1110 are essential for the following structural and functional research of CHU_1110.

Chemical shift assignments of Mb1858 (24-155), a FHA domain-containing protein from Mycobacterium bovis.

The forkhead-associated (FHA) domain is known as a phosphopeptide recognition domain embedded in regulatory proteins from both eukaryotes and bacteria with various biological functions. In this study, the gene encoding a predicted FHA domain from Mb1858 (residues V24-D155 from the 162 amino acid protein Mb1858) in Mycobacterium bovis was cloned, and U-13C/15N-labeled protein was prepared for backbone and side chain resonance assignments by NMR spectroscopy. These assignments are preliminary work towards the determination of the structure and phosphopeptide-binding properties using NMR methods, which will provide useful information about the function of Mb1858 protein.

Chemical shift assignments of polyketide cyclase_like protein CGL2373 from Corynebacterium glutamicum.

Protein CGL2373 from Corynebacterium glutamicum, which is 155 amino acids long and 17.7 kDa, is a member of the polyketide_cyc2 family. As a potential polyketide cyclase, it may play an important role in the biosynthesis of aromatic polyketides that are the source of many bioactive molecules. Here we report the complete 1H, 13C and 15N chemical shift assignments of CGL2373, which lays a foundation for further structural and functional research.

1H, 15N and 13C resonance assignments and secondary structure of PulG, the major pseudopilin from Klebsiella oxytoca type 2 secretion system.

Bacteria use complex transporters to secrete functionally relevant proteins to the extracellular medium. The type 2 secretion system (T2SS) translocates folded proteins involved in bacterial nutrient acquisition, virulence and adaptation. The T2SS pseudopilus is a periplasmic filament, assembled by the polymerization of PulG subunits, the major pseudopilin. Pseudopilin proteins have a conserved N-terminal hydrophobic segment followed by a more variable C-terminal periplasmic and globular domain. To better understand the mechanism of assembly and function of the T2SS, we have been studying the structure and dynamics of PulG by NMR, as well as its interaction with other components of the secretion machinery. As a first step on this study, here we reported the chemical shift assignments of PulG C-terminal domain and its secondary structure prediction based on NMR data.

Interpolating and extrapolating with hmsIST: seeking a tmax for optimal sensitivity, resolution and frequency accuracy.

Non-Uniform Sampling has the potential to exploit the optimal resolution of high-field NMR instruments. This is not possible in 3D and 4D NMR experiments when using traditional uniform sampling due to the long overall measurement time. Nominally, uniformly sampled time domain data acquired to a maximum evolution time tmax can be extended to high resolution via a virtual maximum evolution time t*max while extrapolating with linear prediction or iterative soft thresholding (IST). At the high resolution obtainable with extrapolation of US data, however, the accuracy of peak positions is compromised as observed when comparing inter- and intra-residue peaks in a 3D HNCA experiment. However, the accuracy of peak positions is largely improved by spreading the same number of acquired time domain data points non-uniformly over a larger evolution time to an optimal tmax followed by extrapolation to a total t*max and processing the data with an appropriate reconstruction method, such as hmsIST. To explore the optimum value of experimentally measured tmax to be reached non-uniformly with a given number of sampling points we have created test situations of time-equivalent experiments and evaluate sensitivity and accuracy of peak positions. Here we use signal-to-maximum-noise ratio as the decisive measure of sensitivity. We find that both sensitivity and resolution are optimal when PoissonGap sampling to a tmax of about ½*T2*. Digital resolution is further enhanced by extrapolating the range of acquired time domain data to 2*T2* but without measuring experimental points beyond ½*T2*.

Application of a computer-assisted structure elucidation program for the structural determination of a new terpenoid aldehyde with an unusual skeleton.

The structure of a novel compound from Adansonia digitata has been elucidated, and its 1 H and 13 C NMR spectra have been assigned employing a variety of one-dimensional and two-dimensional NMR techniques without degradative chemistry. The Advanced Chemistry Development ACD/Structure Elucidator software was important for determining part of this structure that contained a fused bicyclic system with very few hydrogen atoms, which in turn, exhibited essentially no discriminating HMBC connectivities throughout that portion of the molecule. Copyright © 2016 John Wiley & Sons, Ltd.

Probabilistic validation of protein NMR chemical shift assignments.

Data validation plays an important role in ensuring the reliability and reproducibility of studies. NMR investigations of the functional properties, dynamics, chemical kinetics, and structures of proteins depend critically on the correctness of chemical shift assignments. We present a novel probabilistic method named ARECA for validating chemical shift assignments that relies on the nuclear Overhauser effect data . ARECA has been evaluated through its application to 26 case studies and has been shown to be complementary to, and usually more reliable than, approaches based on chemical shift databases. ARECA is available online at http://areca.nmrfam.wisc.edu/.

Chemical shift assignments for the Ig2 domain of human obscurin A.

The giant sarcomeric protein obscurin (~720 kDa) is an essential contributor to myofibrillogenesis and acts as the only known tether between the contractile apparatus and the surrounding membrane structures in myofibrils. Genomic characterization of OBSCN suggests a modular architecture, consisting of dozens of individually-folded Ig-like and FnIII-like domains arranged in tandem. Here we describe the sequence-specific chemical shift assignments of the second putative obscurin Ig-like domain (Ig2). This domain specifically binds to MyBP-C slow variant-1 through an unknown mechanism. Ultimately, the assignments presented here will facilitate high-resolution structure determination of Ig2 and provide insight into the specificity of the obscurin-MyBP-C interaction.

1H, 15N and 13C 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 (1)H, (15)N and (13)C chemical shift assignments of (15)N/(13)C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, Cα, Hα, C and side chain Cß assignments. These results can provide the basis for further structural exploration of lcFABP.

Sequential backbone resonance assignments of the E. coli dihydrofolate reductase Gly67Val mutant: folate complex.

Occasionally, a mutation in an exposed loop region causes a significant change in protein function and/or stability. A single mutation Gly67Val of E. coli dihydrofolate reductase (DHFR) in the exposed CD loop is such an example. We have carried out the chemical shift assignments for H(N), N(H), C(α) and C(ß) atoms of the Gly67Val mutant of E. coli DHFR complexed with folate at pH 7.0, 35 °C, and then evaluated the H(N), N(H), C(α) and C(ß) chemical shift changes caused by the mutation. The result indicates that, while the overall secondary structure remains the same, the single mutation Gly67Val causes site-specific conformational changes of the polypeptide backbone restricted around the adenosine-binding subdomain (residues 38-88) and not in the distant catalytic domain.

Chemical shift assignments for S. cerevisiae Ubc13.

The ubiquitination pathway controls several human cellular processes, most notably protein degradation. Ubiquitin, a small signaling protein, is activated by the E1 activating enzyme, transferred to an E2 conjugating enzyme, and then attached to a target substrate through a process that can be facilitated by an E3 ligase enzyme. The enzymatic mechanism of ubiquitin transfer from the E2 conjugating enzyme onto substrate is not clear. The highly conserved HPN motif in E2 catalytic domains is generally thought to help stabilize an oxyanion intermediate formed during ubiquitin transfer. However recent work suggests this motif is instead involved in a structural, non-enzymatic role. As a platform to better understand the E2 catalyzed ubiquitin transfer mechanism, we determined the chemical shift assignments of S. cerevisiae E2 enzyme Ubc13.

Perspectives in magnetic resonance: NMR in the post-FFT era.

Multi-dimensional NMR spectra have traditionally been processed with the fast Fourier transformation (FFT). The availability of high field instruments, the complexity of spectra of large proteins, the narrow signal dispersion of some unstructured proteins, and the time needed to record the necessary increments in the indirect dimensions to exploit the resolution of the highfield instruments make this traditional approach unsatisfactory. New procedures need to be developed beyond uniform sampling of the indirect dimensions and reconstruction methods other than the straight FFT are necessary. Here we discuss approaches of non-uniform sampling (NUS) and suitable reconstruction methods. We expect that such methods will become standard for multi-dimensional NMR data acquisition with complex biological macromolecules and will dramatically enhance the power of modern biological NMR.