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Serber Z Straub W Corsini L Nomura AM Shimba N Craik CS Ortiz de Montellano P Dötsch V 《Journal of the American Chemical Society》2004,126(22):7119-7125
Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [(13)C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs. 相似文献
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Dr. Christina I. Schroeder Dr. Lachlan D. Rash Xavier Vila‐Farrés Dr. K. Johan Rosengren Dr. Mehdi Mobli Prof. Glenn F. King Prof. Paul F. Alewood Prof. David J. Craik Dr. Thomas Durek 《Angewandte Chemie (International ed. in English)》2014,53(4):1017-1020
Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid‐sensing ion channels (ASICs). The 57‐residue polypeptide mambalgin‐2 (Ma‐2) was synthesized by using a combination of solid‐phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three‐finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma‐2 on wild‐type and mutant ASIC1a receptors allowed us to identify α‐helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma‐2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure–activity relationship (SAR) studies and further development of this promising analgesic peptide. 相似文献
5.
Racemic and Quasi‐Racemic X‐ray Structures of Cyclic Disulfide‐Rich Peptide Drug Scaffolds
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Conan K. Wang Gordon J. King Susan E. Northfield Paola G. Ojeda Prof. Dr. David J. Craik 《Angewandte Chemie (International ed. in English)》2014,53(42):11236-11241
Cyclic disulfide‐rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X‐ray structures of these peptides to assist in drug design applications, but disulfide‐rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L ‐ and D ‐forms of three prototypic cyclic disulfide‐rich peptides: SFTI‐1 (14‐mer with one disulfide bond), cVc1.1 (22‐mer with two disulfide bonds), and kB1 (29‐mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Å to 1.9 Å. Additionally, we obtained the quasi‐racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Å and 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides. 相似文献
6.
Plínio C. Sathler Charles S. Craik Toshihiko Takeuchi Russolina B. Zingali Helena C. Castro 《Applied biochemistry and biotechnology》2010,160(8):2355-2365
Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological
potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes
in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying
enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type
plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R,
M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line
and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized
PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland
lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when
ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin
variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization
of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous
proteins in different biological processes. 相似文献
7.
R.J.M. Craik 《Applied Acoustics》1982,15(4):275-282
Conventional methods for determining the properties of building materials by the measurement of the longitudinal wavespeed suffer from the disadvantage that the measurements can only be carried out in localised parts of the building, usually through the thickness of the wall. The techniques, although non-destructive, may mark decorative finishes.Similar methods have been used to obtain the wavespeed from low frequency pulses which may be used in all parts of the structure without damage. The technique is very simple and the results are accurate. The simplicity of the technique makes the method suitable for many applications. 相似文献
8.
D.J. Craik 《Physics letters. A》1983,96(6):283-285
The finite-difference Laplace equation applying at a magnetic interface has been derived by Binns and Lawrenson using the rather difficult concept of “fictitious potential”. The equation may be verified by recourse to a simple model, and this model may further be used to give an alternative and somewhat clearer derivation. 相似文献
9.
It has recently been shown that for situations in which two rooms are connected by a ventilation duct, the principal acoustic path from one room to the adjacent room is that via the duct cavity. As existing theories were found to be inadequate in predicting this path noise reduction, experiments have been carried out to determine the values of the unknown variables. These are described and a design guide for determining the overall noise reduction of the system has been produced. 相似文献
10.
A new, vectorial approach to fast correlation attacks on binary memoryless combiners is proposed. Instead of individual input sequences or their linear combinations, the new attack is targeting subsets of input sequences as a whole thus exploiting the full correlation between the chosen subset and the output sequence. In particular, the set of all the input sequences can be chosen as the target. The attack is based on a novel iterative probabilistic algorithm which is also applicable to general memoryless combiners over finite fields or finite rings. To illustrate the effectiveness of the introduced approach, experimental results obtained for random balanced combining functions are presentedMost of this work was done while he was with Rome CryptoDesign Center, Gemplus, Italy 相似文献