Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N-1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure

AbstractCB2, a CNBr peptide of 36 residues from type I collagen α1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons (1JNH=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (Δδ/ΔT of −3.6 ppb/°C) suggest that the H atoms of the protonated guanidinium moiety of Arg9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354–7359), similar to CB2, our data could indicate that the guanidinium group of Arg9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the Nϵ hydrogen, leaving the four Nη hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric α1(I) CB2 triple helix. The behaviour of Arg9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17°C

EF Loop Conformational Change Triggers Ligand Binding in β-Lactoglobulins

Beta-lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of beta-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of beta-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu89 protonation. The alkaline shift observed for Glu89 pKa in porcine beta-lactoglobulin (pKa 9.7) with respect to the bovine species (pKa 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine beta-lactoglobulin seems to be functional to lipases activity. Theoretical pKa calculations extended to representative beta-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all beta-lactoglobulins

Central nervous system trans-synaptic effects of acute axonal injury : a 1-H magnetic resonance spectroscopy study

N-acetylaspartate (NAA) has previously been proposed as a neuronal marker. 1H magnetic resonance spectroscopy (MRS) is able to detect NAA in brain, and decreases of NAA have been documented after brain injury. The reason for this decrease is not fully understood and neuron loss damage and "dysfunction" have all been proposed. It is hypothesized that acute central nervous system (CNS) deafferentation causes a trans-synaptic NAA decrease and that high resolution 1H MRS is able to detect such a decrease. To test this hypothesis, an experimental model was used in which axonal lesions were obtained by stretch injury in guinea pig right optic nerve (95-99% crossed fibers). The trans-synaptic concentration of NAA, total creatine (Cr), and the NAA/Cr ratio in lateral geniculate bodies (LGB) and superior colliculi (SC) sample extracts were measured 72 h later by high resolution 1H MRS. In the left LGB/SC, which is where right optic nerve fibers project, reductions of NAA and NAA/Cr were found whereas Cr levels were normal. NAA, NAA/Cr, and Cr values were all normal in the right LGB/SC. Histology and EM findings revealed no abnormalities. At 7 days, left LGB/SC NAA and NAA/Cr values were in the normal range. It was concluded that 1) acute deafferentation in the CNS causes a trans-synaptic decrease of NAA levels that can be detected by 1H MRS and 2) NAA decrease may be due to changes of NAA metabolism caused by functional neuronal inactivity rather than neuronal loss, injury or "dysfunction." 1H MRS is a potential tool for the study of functional effect of CNS lesions in vivo

Ethylene-norbornene copolymer microstructure. Assessment and advances based on assignments of 13C NMR spectra

International audienc

Ethylene-norbornene copolymer microstructure. Assessment and advances based on assignments of 13C NMR spectra

International audienc