HNHC: a triple resonance experiment for correlating the H2, N1(N3) and C2 resonances in adenine nucleobases of <Superscript>13</Superscript>C-, <Superscript>15</Superscript>N-labeled RNA oligonucleotides

A novel NMR pulse sequence has been developed that correlates the H2 resonances with the C2 and the N1 (N3) resonances in adenine nucleobases of ¹³C, ¹⁵N labeled oligonucleotides. The pulse scheme of the new 3D-HNHC experiment is composed of a ²J-¹⁵N-HSQC and a ¹J-¹³C-HSQC and utilizes large ²J(H2, N1(N3)) and ¹J(H2, C2) couplings. The experiment was applied to a medium-size ¹³C, ¹⁵N-labeled 36mer RNA. It is useful to resolve assignment ambiguities occurring especially in larger RNA molecules due to resonance overlap in the ¹H-dimension. Therefore, the missing link in correlating the imino H3 resonances of the uracils across the AU base pair to the H8 resonances of the adenines via the novel pulse sequence and the TROSY relayed HCCH-COSY (Simon et al. in J Biomol NMR 20:173–176 2001) is provided. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conformational heterogeneity in the Hsp70 chaperone‐substrate ensemble identified from analysis of NMR‐detected titration data

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl‐TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR‐based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1‐DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes.     

NMR characterization of the conformational fluctuations of the human lymphocyte function‐associated antigen‐1 I‐domain

Lymphocyte function‐associated antigen‐1 (LFA‐1) is an integrin protein that transmits information across the plasma membrane through the so‐called inside‐out and outside‐in signaling mechanisms. To investigate these mechanisms, we carried out an NMR analysis of the dynamics of the LFA‐1 I‐domain, which has enabled us to characterize the motions of this domain on a broad range of timescales. We studied first the internal motions on the nanosecond timescale by spin relaxation measurements and model‐free analysis. We then extended this analysis to the millisecond timescale motions by measuring ¹⁵N‐¹H residual dipolar couplings of the backbone amide groups. We analyzed these results in the context of the three major conformational states of the I‐domain using their corresponding X‐ray crystallographic structures. Our results highlight the importance of the low‐frequency motions of the LFA‐1 I‐domain in the inactive apo‐state. We found in particular that α‐helix 7 is in a position in the apo‐closed state that cannot be fully described by any of the existing X‐ray structures, as it appears to be in dynamic exchange between different conformations. This type of motion seems to represent an inherent property of the LFA‐1 I‐domain and might be relevant for controlling the access to the allosteric binding pocket, as well as for the downward displacement of α‐helix 7 that is required for the activation of LFA‐1.     

Characterization of the Site‐Specific Acid‐Base Equilibria of 3‐Nitrotyrosine

The complete macro‐ and microequilibrium analyses of 3‐nitrotyrosine, a biomarker of oxidative stress damage, are presented for the first time. The protonation macroconstants were determined by ¹H‐NMR‐pH titration, while microconstants were elucidated by a combination of deductive and NMR methods, in which properties of the methyl ester derivative as an auxiliary compound were also studied. Combination of the NMR‐pH characterization of the title and auxiliary compounds and the pair‐interactivity parameters of 3‐iodotyrosine provided the sufficient system to evaluate all the microconstants. NMR‐pH profiles, macroscopic and microscopic protonation schemes, and species‐specific distribution diagrams are included. The phenolate basicity of 3‐nitrotyrosine is 500 times below that of tyrosine, and it is even lower than that of 3‐iodotyrosine. This phenomenon can be explained by the stronger electron withdrawing and the negative mesomeric effect of the nitro group. Based on our results, 89 % of the phenolic OH groups are deprotonated in 3‐NT molecules at the pH of the blood plasma.     

“Setting paint” analogy for the hydrophobic self‐association of tropoelastin into elastin‐like hydrogel

Alkaline tropoelastin solutions (pH 11) were optically clear at low temperatures, but a firm gel formed when the temperature was raised to 37°C. Reversion to a clear solution took place if the temperature was lowered to below 20°C within less than 2 h, but not if 37°C was maintained for several hours. The precipitated elastin‐like hydrogel thus formed did not visually redissolve at low temperatures. Tropoelastin hydrogel was stable to subsequent washings with alkaline solution at 37°C, but at 4°C some hydrogel redissolved showing that association is at least partly reversible. Washing the hydrogel with neutral 8M urea solution at 4°C dissolved less than 10% of tropoelastin in 24 h. We characterized this phenomenon by combining temperature‐controlled light microscopy analysis, ¹H NMR spectroscopy (temperature, diffusion, and relaxation time studies), and UV‐absorption‐based concentration measurements. The self‐association of tropoelastin at pH 11 is due to hydrophobic interactions in an emulsion‐like system in which the spherules coalesce in a manner like a water‐based latex paint that forms a durable hydrophobic sheet as water and the organic solvent evaporate. In the present case, the sedimentation and entanglement of the tropoelastin porous sheets means that reverse dissolution is a kinetically slow process. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 321–330, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A completed structure of the O-polysaccharide from <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> type 10

The structure of the O-specific polysaccharide from Shigella dysenteriae type 10, which has been reported previously in Bioorganic chemistry (1977, vol.3, pp. 1219–1225), is refined: →2)-β-D-Manp-(1→3)-α-D-ManpNAc-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→.