scholarly journals Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling

ChemBioChem ◽  
2019 ◽  
Vol 20 (5) ◽  
pp. 659-666 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan
2018 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.


2011 ◽  
Vol 3 (1) ◽  
pp. 40-49 ◽  
Author(s):  
Flora I. Hinz ◽  
Daniela C. Dieterich ◽  
David A. Tirrell ◽  
Erin M. Schuman

1980 ◽  
Vol 192 (2) ◽  
pp. 719-723 ◽  
Author(s):  
P J Garlick ◽  
M A McNurlan ◽  
V R Preedy

A rapid procedure for measuring the specific radioactivity of phenylalanine in tissues was developed. This facilitates the accurate determination of rates of protein synthesis in a wide range of tissues by injection of 150 mumol of L-[4-(3)H]phenylalanine/100 g body wt. The large dose of amino acid results in a rapid rise in specific radioactivity of free phenylalanine in tissues to values close to that in plasma, followed by a slow but linear fall. This enables the rate of protein synthesis to be calculated from measurements of the specific radioactivity of free and protein-bound phenylalanine in tissues during a 10 min period after injection of radioisotope.


2021 ◽  
Vol 854 (1) ◽  
pp. 012082
Author(s):  
M Sefer ◽  
R B Petronijevic ◽  
D Trbovic ◽  
J Ciric ◽  
T Baltic ◽  
...  

Abstract Amino acids are fundamental for animal nutrition. Their presence is necessary to maintain the normal structure and function of the intestine, and they are key in regulating metabolic pathways for improving health, survival, growth, development, lactation, and reproduction. The animal feed industry invests great resources and efforts to obtain optimal formulations in which the composition of amino acids plays a key role. In support of these aspirations in recent decades, much attention has been paid to the development and improvement of analytical techniques for the reliable, rapid and accurate determination of amino acid content in animal feed. This paper outlines different methodologies for the analysis of amino acid content in animal feed. Various methods, based on different analytical techniques, are presented for determination of amino acids in feed for nutritional and regulatory purposes.


2005 ◽  
Vol 88 (3) ◽  
pp. 877-887 ◽  
Author(s):  
Robert W Peace ◽  
G Sarwar Gilani

Abstract Amino acids in foods exist in a free form or bound in peptides, proteins, or nonpeptide bonded polymers. Naturally occurring L-amino acids are required for protein synthesis and are precursors for essential molecules, such as co-enzymes and nucleic acids. Nonprotein amino acids may also occur in animal tissues as metabolic intermediates or have other important functions. The development of bacterially derived food proteins, genetically modified foods, and new methods of food processing; the production of amino acids for food fortification; and the introduction of new plant food sources have meant that protein amino acids and amino acid enantiomers in foods can have both nutritional and safety implications for humans. There is, therefore, a need for the rapid and accurate determination of amino acids in foods. Determination of the total amino acid content of foods requires protein hydrolysis by various means that must take into account variations in stability of individual amino acids and resistance of different peptide bonds to the hydrolysis procedures. Modern methods for separation and quantitation of free amino acids either before or after protein hydrolysis include ion exchange chromatography, high performance liquid chromatography (LC), gas chromatography, and capillary electrophoresis. Chemical derivatization of amino acids may be required to change them into forms amenable to separation by the various chromatographic methods or to create derivatives with properties, such as fluorescence, that improve their detection. Official methods for hydrolysis and analysis of amino acids in foods for nutritional purposes have been established. LC is currently the most widely used analytical technique, although there is a need for collaborative testing of methods available. Newer developments in chromatographic methodology and detector technology have reduced sample and reagent requirements and improved identification, resolution, and sensitivity of amino acid analyses of food samples.


1988 ◽  
Vol 71 (6) ◽  
pp. 1172-1175
Author(s):  
Ghulam Sarwar ◽  
Herbert G Botting ◽  
Robert W Peace

Abstract The amino acid analysis method using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography was modified for accurate determination of methionine (as methionine sulfone), cysteine/cystine (as cysteic acid), and all other amino acids, except tryptophan, in hydrolyzed samples of foods and feces. A simple liquid chromatographic method (requiring no derivatization) for the determination of tryptophan in alkaline hydrolysates of foods and feces was also developed. Separation of all amino acids by liquid chromatography was completed in 12 min compared with 60-90 min by ion-exchange chromatography. Variation expressed as coefficients of variation (CV) for the determination of most amino acids in the food and feces samples was not more than 4%, which compared favorably with the reproducibility of ion-exchange methods. Data for amino acids and recoveries of amino acid nitrogen obtained by liquid chromatographic methods were also similar to those obtained by conventional ion-exchange procedures.


Author(s):  
R.D. Leapman ◽  
P. Rez ◽  
D.F. Mayers

Microanalysis by EELS has been developing rapidly and though the general form of the spectrum is now understood there is a need to put the technique on a more quantitative basis (1,2). Certain aspects important for microanalysis include: (i) accurate determination of the partial cross sections, σx(α,ΔE) for core excitation when scattering lies inside collection angle a and energy range ΔE above the edge, (ii) behavior of the background intensity due to excitation of less strongly bound electrons, necessary for extrapolation beneath the signal of interest, (iii) departures from the simple hydrogenic K-edge seen in L and M losses, effecting σx and complicating microanalysis. Such problems might be approached empirically but here we describe how computation can elucidate the spectrum shape.The inelastic cross section differential with respect to energy transfer E and momentum transfer q for electrons of energy E0 and velocity v can be written as


Author(s):  
M.A. Gribelyuk ◽  
M. Rühle

A new method is suggested for the accurate determination of the incident beam direction K, crystal thickness t and the coordinates of the basic reciprocal lattice vectors V1 and V2 (Fig. 1) of the ZOLZ plans in pixels of the digitized 2-D CBED pattern. For a given structure model and some estimated values Vest and Kest of some point O in the CBED pattern a set of line scans AkBk is chosen so that all the scans are located within CBED disks.The points on line scans AkBk are conjugate to those on A0B0 since they are shifted by the reciprocal vector gk with respect to each other. As many conjugate scans are considered as CBED disks fall into the energy filtered region of the experimental pattern. Electron intensities of the transmitted beam I0 and diffracted beams Igk for all points on conjugate scans are found as a function of crystal thickness t on the basis of the full dynamical calculation.


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