Conformational selection of syn-cAMP upon binding to the cAMP receptor protein A 1H NMR study

3',5'-Cyclic AMP (cAMP) receptor protein (CRP) is a dimer of apparently identical subunits, each of M r = 22 500 [1,2], which mediates control of catabo-lite-sensitive operons in Escherichia coli [3,4], cAM

Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles. © 2012 Meng et al

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

\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

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