NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 α‐Xylosidase

The study of the interaction of glycoside hydrolases with their substrates is fundamental to diverse applications in medicine, food and feed production, and biomass‐resource utilization. Recent molecular modeling of the α‐xylosidase CjXyl31A from the soil saprophyte Cellvibrio japonicus, together with protein crystallography and enzyme‐kinetic analysis, has suggested that an appended PA14 protein domain, unique among glycoside hydrolase family 31 members, may confer specificity for large oligosaccharide fragments of the ubiquitous plant polysaccharide xyloglucan (J. Larsbrink, A. Izumi, F. M. Ibatullin, A. Nakhai, H. J. Gilbert, G. J. Davies, H. Brumer, Biochem. J. 2011 , 436, 567–580). In the present study, a combination of NMR spectroscopic techniques, including saturation transfer difference (STD) and transfer NOE (TR‐NOE) spectroscopy, was used to reveal extensive interactions between CjXyl31A active‐site variants and xyloglucan hexa‐ and heptasaccharides. The data specifically indicate that the enzyme recognizes the entire cello‐tetraosyl backbone of the substrate and product in positive enzyme subsites and makes further significant interactions with internal pendant α‐(1→6)‐linked xylosyl units. As such, the present analysis provides an important rationalization of previous kinetic data on CjXyl31A and unique insight into the role of the PA14 domain, which was not otherwise obtainable by protein crystallography.     

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Provided that ¹³C‐detected NMR experiments are either preferable or complementary to ¹H detection, we report here tools to determine C^α? C′, C′? N, and C^α? H^α residual dipolar couplings on the basis of the CON experiment. The coupling constants determined on ubiquitin are consistent with the subset measured with the ¹H‐detected HNCO sequences. Since the utilization of residual dipolar couplings may depend on the mobility of the involved nuclei, we also provide tools to measure longitudinal and transverse relaxation rates of N and C′. This new set of experiments is a further development of a whole strategy based on ¹³C direct‐detection NMR spectroscopy for the study of biological macromolecules.     

The Role of Ligand Topology in the Decomplexation of Luminescent Lanthanide Complexes by Dipicolinic Acid

In this study, we present the aqueous solution behavior of two luminescent lanthanide antenna complexes (Eu³⁺? 1 , Dy³⁺? 9 ) with different ligand topologies in the presence of dipicolinic acid (DPA, pyridine‐2,6‐dicarboxylic acid). Macrocyclic (1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic acid, DO3A, 9 ) and acyclic (1,4,7‐triazaheptane‐1,1,7,7‐tetraacetic acid, DTTA, 1 ) ligands have been selected to form a ratiometric pair in which Dy³⁺? 9 acts as a reference and Eu³⁺? 1 acts as a probe for the recognition of DPA. The pair of luminescent complexes in water reveals the capability to work as a DPA luminescent sensor. The change of emission intensity of Eu³⁺ indicates the occurrence of a new sensitization path for the lanthanide cation through excitation of DPA. NMR evidence implies the presence of free 1 and mass spectrometry shows the formation of emitting [EuDPA₂]^? as a result of a ligand exchange reaction.     

Grafting amount and structural characteristics of microcrystalline cellulose functionalized with different aminosilane contents

Cellulose - Microcrystalline cellulose (MCC) has unique properties and its use as reinforcement for polymer composites has been increasing. However, the intrinsic incompatibility with most polymers...     

Halogen Bonding Assembles Anion⋅⋅⋅Anion Architectures in Non-centrosymmetric Iodate and Bromate Crystals

Single crystal X-ray diffraction of iodate and bromate salts shows that the I and Br atoms in IO₃⁻ and BrO₃⁻ anions form short and linear O−I/Br ⋅⋅⋅ O contacts with the O atoms of nearby anions. Non-centrosymmetric systems are formed wherein anions are orderly aligned into supramolecular 1D and 2D networks. Theoretical evidences, namely the outcome of QTAIM and NCIplot studies, prove the attractive nature of these contacts and the ability of iodate and bromate anions to act as robust halogen bond (HaB) donors. The HaB is proposed as a general and effective assisting tool to control the architecture of acentric iodate salts.     

Surface modification of proteins by covalent binding of acrylic polymers

Surface modification of enzymes for a potential use in therapy was obtained with a new type of tailor-made copolymers ofNacryloylmorpholine andN-acryloxysuccinimide. The first monomer was designed to confer solubility on the polymer, whereas the second was used to give it reactivity toward protein amino groups. The reactivity of polymers of different composition towards amino acid derivatives and model proteins, such as catalase and ribonuclease-A, is described. Water soluble and catalytically active enzyme derivatives were obained using copolymers prepared with a mixture of N-acryloxysuccinimide andn-acryloylmorpholine in a 1:99 molar ratio. At increasing molar ratio (3:97, 10:90) extensive crosslinking between polymer and enzymes takes place, yielding insoluble adducts.     

Chemical basis of peptidoglycan discrimination by PrkC, a key kinase involved in bacterial resuscitation from dormancy

Bacterial Ser/Thr kinases modulate a wide number of cellular processes. In Bacillus subtilis , the Ser/Thr kinase PrkC has been shown to induce germination of bacterial spores in response to DAP-type but not Lys-type cell wall muropeptides. Muropeptides are a clear molecular signal that growing conditions are promising, since they are produced during cell wall peptidoglycan remodeling associated with cell growth and division of neighboring bacteria. However, whether muropeptides are able to bind the protein physically and how the extracellular region is able to distinguish the two types of muropeptides remains unclear. Here we tackled the important question of how the extracellular region of PrkC (EC-PrkC) senses muropeptides. By coupling NMR techniques and protein mutagenesis, we exploited the structural requirements necessary for recognition and binding and proved that muropeptides physically bind to EC-PrkC through DAP-moiety-mediated interactions with an arginine residue, Arg500, belonging to the protein C-terminal PASTA domain. Notably, mutation of this arginine completely suppresses muropeptide binding. Our data provide the first molecular clues into the mechanism of sensing of muropeptides by PrkC.