Labeling residue-specific

Figure 5.18 (A) COST versus the RMSD (A) to the target pose for CDK2/4. The predicted protein chemical shifts were set to the corresponding BMRB average values. The 3D X-filtered NOESY spectrum was filtered to simulate data that could be extracted from residue-specific labeled protein. (B) Superposition of target pose and the minimum cost pose (dark gray) from (A).

Fig. 2.T. [1BN, 1H]-HSQC spectrum of 1BN-labeled recombinant goat a-lactalbumin at pH 6.3 and 25°C in 95% H20/5% D20 (Nakamura et al., unpublished). Peaks are labeled with their residue-specific assignments...

Salopek-Sondi, B. and Luck, L. A. (2002) 19F NMR study of the leucine-specific binding protein of Escherichia coli mutagenesis and assignment of the 5-fluorotryptophan-labeled residues. Protein Engineering, 15, 855-859. 

Figure 18 ABPP probe for glycosidase labeling, (a) Specific probe design using sialic acid as recognition head and a quinone methide as reactive group precursor, (b) Upon activation by glycosidase-mediated cleavage of sialic acid, a reactive quinone methide intermediate is generated that rapidly reacts with residues in the active site.

TM) protein bacteriorhodopsin (bR) in oriented purple membranes. Our initial numerical studies indicated that the spectra from the two types of 2D experiments on uniformly N labeled bR would be too crowded to allow for assignment of all resonances.For this reason, our first experiments focused on samples with more diluted labeling patterns, specifically bR with N labeled methionines, i.e, Met-bR. Figure 13a shows the sequence and the tentative location of the secondary stmcture elements of bR highlighting the nine Met residues of which six or seven are expected to be in the helix region (Fig. 13b). The experimental PISEMA and HETCOR... 

All detection so far in microbial ecology has been with 32p-iabeled probes. Because of the radiation exposure and short shelf-life of these probes, non-radioactive probes would be of great value Such probes have been developed, e.g., the biotin-labeled residues which yield a color reaction, but they have not yet proved sensitive or specific enough to generate any interest by microbial ecologists. 

The NADP-specific enzyme of Neurospora contains a lysine residue with pK = 7.6 at 34°, which reacts with pyridoxal phosphate or iV-ethylmaleimide to yield an inactive enzyme (65). The heat of ionization of the -amino group as judged by its reaction with A -ethylmale-imide is 12,6 2.0 kcal/mole. The labeled residue, Lys-113, is in a homologous sequence with that of the vertebrate enzymes (Fig. 5). 

Noncompetitive assays can only be apphed for high-molecular-mass analytes with more than one antigenic determinant (i.e. Ag) or low-molecular-mass analytes (haptens) bound to a solid phase, exposing the antigenic determinant. They work with an Ab excess. Noncompetitive lAs have been employed for the detection of soil-bound pesticides. In this case the soil particles, to which the pesticide residues have bound, form the sohd phase, and the residues can be detected by a labeled Ab specific to the analyte. 

Eighteen site-directed mutants of bacteriorhodopsin were prepared, each with a single X cysteine (Cys) substitution sequentially placed from positions 125 to 142. These residues are in a region of the protein which starts near the end of one transmembrane helix, passes through an interhelical loop, and then stretches well into the next helical segment (Fig. 7). The mutants were reacted with methane thiosulfonate spin label, which specifically attaches to the Cys (Fig. 1). Extreme care was taken to characterize the folding and functionality of each spin-labeled mutant to ensure that there was little measurable perturbation from the... 

For the production purpose, abolishment of feedback control and fusion of sequential enzymes may be desirable. The recombinant technology is the method of choice when the redesigning involves substimtions between coded amino acids. However, for substitutions involving artificial, noncoded amino acids or their analogues/derivatives (e.g. post-translationally modified amino acids, isotopic label of specific residue), chemical methods in particular semi-chemical synthesis becomes necessary (Chaiken, 1981). The frag-ments/chains of modified, synthetic or expressed polypeptides are enzymatically or chemically ligated (Muir, 2003). 

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