13C, 2H NMR Studies of Structural and Dynamical Modifications of Glucose-Exposed Porcine Aortic Elastin

Elastin, the principal component of the elastic fiber of the extracellular matrix, imparts to vertebrate tissues remarkable resilience and longevity. This work focuses on elucidating dynamical and structural modifications of porcine aortic elastin exposed to glucose by solid-state NMR spectroscopic and relaxation methodologies. Results from macroscopic stress-strain tests are also presented and indicate that glucose-treated elastin is mechanically stiffer than the same tissue without glucose treatment. These measurements show a large hysteresis in the stress-strain behavior of glucose-treated elastin—a well-known signature of viscoelasticity. Two-dimensional relaxation NMR methods were used to investigate the correlation time, distribution, and population of water in these samples. Differences are observed between the relative populations of water, whereas the measured correlation times of tumbling motion of water across the samples were similar. ¹³C magic-angle-spinning NMR methods were applied to investigate structural and dynamical modifications after glucose treatment. Although some overall structure is preserved, the process of glucose exposure results in more heterogeneous structures and slower mobility. The correlation times of tumbling motion of the ¹³C-¹H internuclear vectors in the glucose-treated sample are larger than in untreated samples, pointing to their more rigid structure. The ¹³C cross-polarization spectra reveal a notably increased α-helical character in the alanine motifs after glucose exposure. Results from molecular dynamics simulations are provided that add further insight into dynamical and structural changes of a short repeat, [VPGVG]₅, an alanine pentamer, desmosine, and isodesmosine sites with and without glucose. The simulations point to changes in the entropic and energetic contributions in the retractive forces of VPGVG and AAAAA motifs. The most notable change is the increase of the energetic contribution in the retractive force due to peptide-glucose interactions of the VPGVG motif, which may play an important role in the observed stiffening in glucose-treated elastin.     

DynBench3D,a Web-Resource to Dynamically Generate Benchmark Sets of Large Heteromeric Protein Complexes

Multi-protein machines are responsible for most cellular tasks, and many efforts have been invested in the systematic identification and characterization of thousands of these macromolecular assemblies. However, unfortunately, the (quasi) atomic details necessary to understand their function are available only for a tiny fraction of the known complexes. The computational biology community is developing strategies to integrate structural data of different nature, from electron microscopy to X-ray crystallography, to model large molecular machines, as it has been done for individual proteins and interactions with remarkable success. However, unlike for binary interactions, there is no reliable gold-standard set of three-dimensional (3D) complexes to benchmark the performance of these methodologies and detect their limitations. Here, we present a strategy to dynamically generate non-redundant sets of 3D heteromeric complexes with three or more components. By changing the values of sequence identity and component overlap between assemblies required to define complex redundancy, we can create sets of representative complexes with known 3D structure (i.e., target complexes). Using an identity threshold of 20% and imposing a fraction of component overlap of < 0.5, we identify 495 unique target complexes, which represent a real non-redundant set of heteromeric assemblies with known 3D structure. Moreover, for each target complex, we also identify a set of assemblies, of varying degrees of identity and component overlap, that can be readily used as input in a complex modeling exercise (i.e., template subcomplexes). We hope that resources like this will significantly help the development and progress assessment of novel methodologies, as docking benchmarks and blind prediction contests did. The interactive resource is accessible at https://DynBench3D.irbbarcelona.org.     

The trans labilization of cis-[PtCl2(13CH3NH2)2] by glutathione can be monitored at physiological pH by [1H,13C] HSQC NMR

In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl₂(¹³CH₃NH₂)₂] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release of ¹³CH₃NH₂ but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds are formed and Pt–NH₃ bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C] HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best method for characterizing the different species in solution. However, the intensity of the peaks in the ¹⁵NH₃–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity, and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum complexes with sulfur-containing ligands.     

Solution conformation of the type I collagen alpha-2 chain telopeptides studied by 1H and 13C NMR spectroscopy

The high-field 1H and 13C NMR studies of the N- and C-terminal telopeptides of the alpha-2 chain of collagen were carried out in CD3OH/H2O solutions. All proton assignments are based on two-dimensional phase-sensitive COSY and ROESY experiments. The conformation of the N-telopeptide (nonamer) is predominantly extended with a small proportion of the molecules existing in a type I beta turn. The four residues involved in this turn are D3-A4-K5-G6 which is stabilized by a C = O(D3)-NH(G6) hydrogen bond. The C-terminal telopeptide is extended throughout. A model is proposed involving charge-charge and hydrophobic interactions between the extended alpha-2 chain N-telopeptide and the adjacent segments of triple-helix. A similar model is proposed for the C-telopeptide.     

Comparison of Protein Structures in Solution Using Local Conformations Derived from NMR Data: Application to Cytochrome c

Abstract

Structural comparisons of proteins in solution are often required to examine structure-functional relationships, study structural effects of mutations or distinguish between various forms of the same molecule under different conditions. A nuclear magnetic resonance (NMR) based probabilistic strategy is presented and used to study the structural differences between the two redox states of cytochrome C in solution. A probabilistic approach is employed to calculate the main chain conformations of horse ferro- and ferricytochrome C in solution, based on the published sequential d connectivity data. Conformational differences between the two oxidation states of horse cytochrome C in solution are found to be statistically significant. The largest changes in conformation are at residues Lys27, Thr28, Leu32, Gln42, Thr47, Tyr48, Thr49, Glu69, Lys72, Met80, Phe82, Ile85 and Lys86, all of which are close to the heme (within 14 Å of the heme iron in the high resolution Xray structure of tuna cytochrome c). We suggest that these conformational changes may modulate local dipole moments and hence influence the interactions of cytochrome C with its physiological redox partners during the electron transfer process. The oxidation state dependent conformational differences are found to be much greater in solution than in the crystalline state, and the solution and crystal structures differ significantly in regions close to the heme. These results suggest that the highly charged nature of cytochrome C makes this protein particularly sensitive to the ionic strength of its environment and leads to differences between crystal and solution structures in the same oxidation state. In such cases, crystal structures must be used with caution for modeling molecular interactions in vivo. More generally, this analysis indicates that the determination of accurate local conformations based on nmr data can provide useful information about structure-functional aspects of proteins in solution.     

Iterative Elastic 3D-to-2D Alignment Method Using Normal Modes for Studying Structural Dynamics of Large Macromolecular Complexes

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Ethanol Extraction Requirement for Purification of Protein Labeled with [3H]Leucine in Aquatic Bacterial Production Studies

The trichloroacetic acid (TCA)-insoluble fraction of water column bacteria labeled with [³H]leucine contained an ethanol-soluble fraction accounting for up to 44% of the label. A component of the ethanol-soluble fraction is [³H]leucine. Labeled-protein purification requires an ethanol wash step. Cold TCA can replace hot TCA for precipitation of labeled proteins.     

Selective `unlabeling' of amino acids in fractionally 13C labeled proteins: An approach for stereospecific NMR assignments of CH3 groups in Val and Leu residues

A novel methodology for stereospecific NMR assignments of methyl (CH₃) groups of Val and Leu residues in fractionally ¹³C-labeled proteins is presented. The approach is based on selective `unlabeling' of specific amino acids in proteins while fractionally <sup>13</sup>C-labeling the rest. A 2D [<sup>13</sup>C-<sup>1</sup>H] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the `unlabeled' amino acid residues. Such spectral simplification aids in unambiguous stereospecific assignment of diastereotopic CH₃ groups in Val and Leu residues in large proteins. This methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP).     

1H, 15N, and 13C NMR chemical shift assignments for the Ig3 domain of palladin

As part of our NMR structure determination of the palladin Ig3 domain, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C, and ¹⁵N nuclei.     

Synthesis of 3H and 13C labeled mRNA cap dinucleotides--useful tools for NMR, biochemical, and biological studies

For deeper understanding the roles of the mRNA cap structure in cellular processes isotopically labeled dinucleotide cap analogues have been synthesized as tools for NMR and in vivo studies. Tritium or carbon C-13 labeled methyl iodide was used as a source of the isotope material. In order to minimize the number of steps during the radioisotopic synthesis the methylation with tritium labeled methyl iodide was performed with Gp(3)G as a substrate. The C-13 isotope was introduced into the cap dinucleotide by methylation of GDP with C-13 Methyl iodide, followed by coupling the product with guanosine 5'-phosphorimidazolide in DMF with zinc chloride as a catalyst.     

Interaction of the Water-Soluble Protein Aprotinin with Liposomes: Gel-Filtration,Turbidity Studies,and 31P NMR Studies

Abstract

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; ³¹P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV–aprotinin complexes was determined. Lipid–protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. ³¹P NMR spectroscopy of the MLV–aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

Solution structure of the calponin CH domain and fitting to the 3D-helical reconstruction of F-actin:calponin

Calponin is involved in the regulation of contractility and organization of the actin cytoskeleton in smooth muscle cells. It is the archetypal member of the calponin homology (CH) domain family of actin binding proteins that includes cytoskeletal linkers such as alpha-actinin, spectrin, and dystrophin, and regulatory proteins including VAV, IQGAP, and calponin. We have determined the first structure of a CH domain from a single CH domain-containing protein, that of calponin, and have fitted the NMR-derived coordinates to the 3D-helical reconstruction of the F-actin:calponin complex using cryo-electron microscopy. The tertiary fold of this single CH domain is typical of, yet significantly different from, those of the CH domains that occur in tandem pairs to form high-affinity ABDs in other proteins. We thus provide a structural insight into the mode of interaction between F-actin and CH domain-containing proteins.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Characterizing ZC3H18, a Multi-domain Protein at the Interface of RNA Production and Destruction Decisions

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