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FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein 下载免费PDF全文
A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall alpha-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage. 相似文献
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VV. R. PHILIPSON F.L.S. 《Botanical journal of the Linnean Society. Linnean Society of London》1987,95(1):19-25
Problems presented by genera, or small groups of genera, which have been given family rank are reviewed, and the genera are divided into a number of categories according to the closeness of their affinity to other genera or families. Satellite genera that stand in close relation to families should be united with them. Binary families, that have been divided into two (or more) related families, should be re–united. Families connected by linking genera, should, logically, be united but practical considerations usually prevent this. Clusters of diverse but more or less distantly related genera present unusual problems, being treated either as several, often monogeneric families or as a loosely structured family. Truly isolated genera must be given family and often ordinal rank. 相似文献
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Additive partial linear models with measurement errors 总被引:1,自引:0,他引:1
Liang Hua; Thurston Sally W.; Ruppert David; Apanasovich Tatiyana; Hauser Russ 《Biometrika》2008,95(3):667-678
We consider statistical inference for additive partial linearmodels when the linear covariate is measured with error. Wepropose attenuation-to-correction and simulation-extrapolation,simex, estimators of the parameter of interest. It is shownthat the first resulting estimator is asymptotically normaland requires no undersmoothing. This is an advantage of ourestimator over existing backfitting-based estimators for semiparametricadditive models which require undersmoothing of the nonparametriccomponent in order for the estimator of the parametric componentto be root-n consistent. This feature stems from a decreaseof the bias of the resulting estimator, which is appropriatelyderived using a profile procedure. A similar characteristicin semiparametric partially linear models was obtained by Wanget al. (2005). We also discuss the asymptotics of the proposedsimex approach. Finite-sample performance of the proposed estimatorsis assessed by simulation experiments. The proposed methodsare applied to a dataset from a semen study. 相似文献
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FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association 下载免费PDF全文
A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state F?rster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems. The applicability of the simulation-based fitting approach was verified on simulated FRET data and then applied to determine the structural properties of the well-known major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants Y24C, G38C, and T46C of this protein were produced and specifically labeled with the fluorescence label AEDANS. The energy transfer data from the natural tryptophan at position 26, which is used as a donor, to AEDANS were analyzed assuming a helix model for the transmembrane domain of the protein. As a result of the FRET data analysis, the topology and bilayer embedment of this domain were quantitatively characterized. The resulting tilt of the transmembrane helix of the protein is 18 +/- 2 degrees. The tryptophan is located at a distance of 8.5 +/- 0.5 A from the membrane center. No specific aggregation of the protein was found. The methodology developed here is not limited to M13 major coat protein and can be used in principle to study the bilayer embedment of any small protein with a single transmembrane domain. 相似文献