Relationships between the Precision of High-Resolution Protein NMR Structures, Solution-Order Parameters, and Crystallographic B Factors

One of the principal motivations for studying proteins by nuclear magnetic resonance stems from the desire to describe the solution structure of these molecules as compared to the generally perceived static picture obtained by X-ray crystallography. Indeed, it is one of the unique features of NMR spectroscopy that in addition to structural data, dynamic properties can be probed and characterized by measuring relaxation parameters. Furthermore, any mobility of the protein in solution will necessarily modulate the measured NMR parameters and should influence the resulting structure. It has been argued that regions of a protein that are highly mobile would be expected to be defined to a lesser degree of precision than regions that are rigid (1. 2 )

Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used a new technique of site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA - an essential chromosomal gene of E. coli encoding dihydrofolate reductase (DHFR) - to determine how changes in protein stability, activity and abundance affect fitness. In total, 27 E.coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance; suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30oC. At 42oC, on the other hand, the picture was mixed, yet remarkable: a few strains carrying mutant DHFR proteins aggregated rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42oC by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation

\u3csup\u3e1\u3c/sup\u3eH, \u3csup\u3e15\u3c/sup\u3eN, \u3csup\u3e13\u3c/sup\u3eC, and \u3csup\u3e13\u3c/sup\u3eCO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of \u3e95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i+l),NH(i+l) correlations are observed in the HNCO experiment, the CαH(i),Cα(i)-N(i+l) correlations in the HCA(CO)N experiment, the Cα(i)-N(i+l),NH(i+ 1) correlations in the HNCA and HN(C0)CA experiments, the CαH(i)-N(i+ l),NH(i+l) correlations in the H(CA)NH and HN(CO)HB experiments, and the Cβ(i)-N(i+ l),NH(i+ 1) correlations in the HN(CO)HB experiments. The backbone intraresidue CαH(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the CβH(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the l3Cα(i)-l5N(i)-NH(i) correlations by the HNCA experiment, and the CαH(1)-13Cα(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution

Silylation of titanium-containing amorphous silica catalyst: effect on the alkenes epoxidation with H2O2

The surface of a Ti/SiO2 catalyst was silylated using hexamethyldisilazane (HMDS) and tetramethyldisilazane (TMDS) as silylating reagents in vapor phase. The silylation of silanol (Si–OH) on the catalysts was confirmed by diffuse reflectance UV-Vis, DRIFT spectroscopy and solid-state 29Si MAS NMR techniques. Silylation with TMDS improves the catalytic performance of Ti/SiO2 catalyst more significantly than with HMDS and this is mainly due to the steric bulk effects of trimethylsilyl groups from HMDS as compared to dimethylsilyl groups from TMDS. The silylation degree with TMDS was found to be higher than with HMDS and no changes in the silylation degree with TMDS was observed for silylation times longer than 2 h. Despite the silylation route employed, the silylated surface is hydrophobic which enhances not only in hydrogen peroxide efficiency but also and in selectivity to epoxide in the epoxidation of alkenes with H2O2. These effects are more evident when the hydrogen peroxide concentration is higher and particularly for the catalyst modified with TMDS.Peer reviewe

Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles

The stability, activity, and solubility of a protein sequence are determined by a delicate balance of molecular interactions in a variety of conformational states. Even so, most computational protein design methods model sequences in the context of a single native conformation. Simulations that model the native state as an ensemble have been mostly neglected due to the lack of sufficiently powerful optimization algorithms for multistate design. Here, we have applied our multistate design algorithm to study the potential utility of various forms of input structural data for design. To facilitate a more thorough analysis, we developed new methods for the design and high-throughput stability determination of combinatorial mutation libraries based on protein design calculations. The application of these methods to the core design of a small model system produced many variants with improved thermodynamic stability and showed that multistate design methods can be readily applied to large structural ensembles. We found that exhaustive screening of our designed libraries helped to clarify several sources of simulation error that would have otherwise been difficult to ascertain. Interestingly, the lack of correlation between our simulated and experimentally measured stability values shows clearly that a design procedure need not reproduce experimental data exactly to achieve success. This surprising result suggests potentially fruitful directions for the improvement of computational protein design technology

Backbone resonance assignments of the monomeric DUF59 domain of human Fam96a

Proteins containing a domain of unknown function 59 (DUF59) appear to have a variety of physiological functions, ranging from iron-sulfur cluster assembly to DNA repair. DUF59 proteins have been found in bacteria, archaea and eukaryotes, however Fam96a and Fam96b are the only mammalian proteins predicted to contain a DUF59 domain. Fam96a is an 18 kDa protein comprised primarily of a DUF59 domain (residues 31-157) and an N-terminal signal peptide (residues 1-27). Interestingly, the DUF59 domain of Fam96a exists as monomeric and dimeric forms in solution, and X-ray crystallography studies of both forms unexpectedly revealed two different domain-swapped dimer structures. Here we report the backbone resonance assignments and secondary structure of the monomeric form of the 127 residue DUF59 domain of human Fam96a. This study provides the basis for further understanding the structural variability exhibited by Fam96a and the mechanism for domain swapping

Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. © 2011 Zhao et al

The Global Dynamics of Inequality (GINI) project: analysing archaeological housing data

The GINI project investigates the dynamics of inequality among populations over the long term by synthesising global archaeological housing data. This project brings archaeologists together from around the world to assess hypotheses concerning the causes and consequences of inequality that are of relevance to contemporary societies globally

Genomic and dietary discontinuities during the Mesolithic and Neolithic in Sicily

Summary Sicily is a key region for understanding the agricultural transition in the Mediterranean, due to its central position. Here, we present genomic and stable isotopic data for 19 prehistoric Sicilians covering the Mesolithic to Bronze Age periods (10,700-4,100 yBP). We find that Early Mesolithic hunter-gatherers (HGs) from Sicily are a highly drifted lineage of the Early Holocene western European HGs, while Late Mesolithic HGs carry ∼20% ancestry related to northern and (south)eastern European HGs, indicating substantial gene flow. Early Neolithic farmers are genetically most similar to farmers from the Balkans and Greece, with only ∼7% ancestry from local Mesolithic HGs. The genetic discontinuities during the Mesolithic and Early Neolithic match changes in material culture and diet. Three outlying individuals dated to ∼8,000 yBP, however, suggest that hunter-gatherers interacted with incoming farmers at Grotta dell’Uzzo, resulting in a mixed economy and diet for a brief interlude at the Mesolithic-Neolithic transition.- Introduction - Results -- Genetically-distinct groups of prehistoric Sicilians -- Genomic and dietary transitions in Sicily during the Mesolithic and Early Neolithic -- Did Sicilian Late Mesolithic foragers adopt some aspects of early farming? - Discussion -- Limitations of the stud

On Side-Chain Conformational Entropy of Proteins

The role of side-chain entropy (SCE) in protein folding has long been speculated about but is still not fully understood. Utilizing a newly developed Monte Carlo method, we conducted a systematic investigation of how the SCE relates to the size of the protein and how it differs among a protein's X-ray, NMR, and decoy structures. We estimated the SCE for a set of 675 nonhomologous proteins, and observed that there is a significant SCE for both exposed and buried residues for all these proteins—the contribution of buried residues approaches ∼40% of the overall SCE. Furthermore, the SCE can be quite different for structures with similar compactness or even similar conformations. As a striking example, we found that proteins' X-ray structures appear to pack more “cleverly” than their NMR or decoy counterparts in the sense of retaining higher SCE while achieving comparable compactness, which suggests that the SCE plays an important role in favouring native protein structures. By including a SCE term in a simple free energy function, we can significantly improve the discrimination of native protein structures from decoys