Hsp33 is a prokaryotic molecular chaperon that exerts a holdase activity upon response to an oxidative stress at raised temperature. In particular, intramolecular disulfide bond formation between the four conserved cysteines that bind a zinc ion in reduced state is known to be critically associated with the redox sensing. Here we report the backbone NMR assignment results of the half-oxidized Hsp33, where only two of the four cysteines form an intramolecular disulfide bond. Almost all of the resolved peaks could be unambiguously assigned, although the total assignments extent reached just about 50%. Majority of the missing assignments could be attributed to a significant spectral collapse, largely due to the oxidation-induced unfolding of the C-terminal redox-switch domain. These results support two previous suggestions: conformational change in the first oxidation step is localized mainly in the C-terminal zinc-binding domain, and the half-oxidized form would be still inactive. However, some additional regions appeared to be potentially changed from the reduced state, which suggest that the half-oxidized conformation would be an intermediate state that is more labile to heat and/or further oxidation.

Helicobacter pylori (H. pylori) survives in acidic and fluctuating pH conditions of the stomach. The pH effect on H. pylori proteins is important for the advanced understanding of its evolution and viability, although this bacterium has the molecular machinery that neutralizes the acidic condition. HP1492 is known as a conserved NifU-like protein from H. pylori. NifU is a nitrogen fixation protein that mediates the transfer of iron-sulfur (Fe-S) cluster to iron-sulfur proteins like ferredoxin. Commonly, the monomeric reduced state of NifU can be converted to the dimeric oxidized state by intermolecular disulfide bond formation. Because it remains unclear that HP1492 actually behaves as known NifU protein, we first found that this protein can adopt both oxidized and reduced forms using size exclusion chromatography. Circular dichroism experiment showed that HP1492 is relatively well-structured at pH 6.5, compared to other pH conditions. On the basis of the backbone resonance assignment of HP1492, we further characterized the residues that are sensitive to pH using NMR spectroscopy. These residues showing large chemical shift changes could be mapped onto the secondary structure of the protein. Our results could provide the foundation for structural and biophysical studies on a wide spectrum of NifU proteins.

Hsp31 protein is one of the members of DJ-1 superfamily proteins and has a dimeric structure of which molecular weight (MW) is 62 kDa. The mutation of DJ-1 is closely related to early onset of Parkinson's disease. Hsp31 displays $Zn^{+2}$-binding activity and was first reported to be a holding chaperone in E. coli. Its additional glyoxalase III active has recently been characterized. Moreover, an incubation at $60^{\circ}C$ induces Hsp31 protein to form a high MW oligomer (HMW) in vitro, which accomplishes an elevated holding chaperone activity. The NMR technique is elegant method to probe any local or global structural change of a protein in responses to environmental stresses (heat, pH, and metal). Although the presence of the backbone chemical shifts (bbCSs) is a prerequisite for detailed NMR analyses of the structural changes, general HSQC-based triple resonance experiments could not be used for 62 kDa Hsp31 protein. Here, we prepared the per-deuterated Hsp31 and performed the TROSY-based triple resonance experiments for the bbCSs assignment. Here, detailed processes of per-deuteration and the NMR experiments are described for other similar NMR approaches.

The thermodynamic properties and structural geometry of $KMgCl_3{\cdot}6H_2O$ were investigated using thermogravimetric analysis, differential scanning calorimetry, and nuclear magnetic resonance. The initial mass loss occurs around 351 K ($=T_d$), which is interpreted as the onset of partial thermal decomposition. Phase transition temperatures were found at 435 K ($=T_{C1}$) and 481 K ($=T_{C2}$). The temperature dependences of the spin-lattice relaxation time $T_1$ for the $^1H$ nucleus changes abruptly near $T_{C1}$. These changes are associated with changes in the geometry of the arrangement of octahedral water molecules.

Hyperpolarization methods are the most emerging techniques in the field of magnetic resonance (MR) researches since they make a contribution to overcoming sensitivity limitation of MR spectroscopy and imaging, leading to new fields of researches, real-time in vivo metabolic/molecular imaging and MR analysis of chemical/biological reactions in non-equilibrium conditions. Make use of enormous signal enrichments, it becomes feasible to investigate various chemical and biochemical systems with low ${\gamma}$ nuclei in real-time. This review deals with the theoretical principals of common hyperpolarization methods and their experimental features. In addition, more detailed theories, mechanisms, and applications of dissolution dynamic nuclear polarization (D-DNP) are discussed.

Nuclear magnetic resonance (NMR) spectroscopy, owing to its ability to provide atomic level information on molecular structure, dynamics and interaction, has become one of the most powerful methods in early drug discovery where hit finding and hit-to-lead generation are mainly pursued. In recent years, drug discovery programs originating from the fragment-based drug discovery (FBDD) strategies have been widely incorporated into academia and industry in which a wide variety of NMR methods become an indispensable arsenal to elucidate the binding of small molecules onto bimolecular targets. In this review, I briefly describe FBDD and introduce NMR methods mainly used in FBDD campaigns of my company. In addition, quality control of fragment library and practical NMR methods in industrial aspect are discussed shortly.

NMR-based structural studies on membrane proteins are appreciated quite challenging due to various reasons, generally including the narrow dispersion of NMR spectra, the severe peak broadening, and the lack of long range NOEs. In spite of the poor biophysical properties, structural studies on membrane proteins have got to go on, considering their functional importance in biological systems. In this review, we provide a simple overview of the techniques generally used in structural studies of membrane proteins by solution NMR, with experimental examples of a helical membrane protein, caveolin 3. Detergent screening is usually employed as the first step and the selection of appropriate detergent is the most important for successful approach to membrane proteins. Various tools can then be applied as specialized NMR techniques in solution that include sample deteuration, amino-acid selective isotope labeling, residual dipolar coupling, and paramagnetic relaxation enhancement.

In some pathogenic bacteria, there are RNA thermometers, which regulate the production of virulence associated factors or heat shock proteins depending on temperature changes. Like a riboswitches, RNA thermometers are located in the 5'-untranslated region and involved translational gene regulatory mechanism. RNA thermometers block the ribosome-binding site and start codon area under the $37^{\circ}C$ within their secondary structure. After bacterial infection, increased the temperature in the host causes conformations changes of RNA, and the ribosome-binding site is exposed for translational initiation. Because structural differences between open and closed forms of RNA thermometers are mainly mediated by base pairing changes, NMR spectroscopy is a very useful method to study these thermodynamically changing RNA structure. In this review, we briefly provide a fundamental function of RNA thermometers, and also suggest a proper NMR experiments for studying RNA thermometers.

Acyl carrier protein is related with fatty acid biosynthesis in which specific enzymes are involved. Especially, acyl carrier protein (ACP) is the key component in the growing of fatty acid chain. ACP is the small, very acidic protein that covalently binds various intermediates of fatty acyl chain. Acylation of ACP is mediated by holo-acyl carrier protein synthase (ACPS), which transfers the 4'PP-moiety of CoA to the 36th residue Ser of apo ACP. Acyl carrier protein P (ACPP) is one of ACPs from Helicobacter plyori. The NMR structure of ACPP consists of four helices, which were reported previously. Here we show how acylation of ACPP can affect the overall structure of ACPP and figured out the contact surface of ACPP to acyl chain attached during expression of ACPP in E. coli. Based on the chemical shift perturbation data, the acylation of ACCP seems to affect the conformation of the long loop connecting helix I and helix II as well as the second short loop connecting helix II and helix III. The significant chemical shift change of Ile 54 upon acylation supports the contact of acyl chain and the second loop.