Proteins labelled protein molecules showing

AMCA-NHS, succinimidyl-7-amino-4-methylcoumarin-3-acetic acid, is an amine-reactive derivative of AMCA containing an NHS ester on its carboxylate group (Thermo Fisher). The result is reactivity directed toward amine-containing molecules, forming amide linkages with the AMCA fluorophore (Figure 9.23). Proteins labeled with AMCA show little-to-no effect on the isoelectric point of the molecule. 

Figure 6.12 Labelling a protein molecule with a donor and acceptor group to show the dependence of FRET on distance...

The results obtained show that the dipole-relaxational motions in protein molecules are really very retarded as compared to such motions in the environment of aromatic molecules dissolved in liquid solvents (where they occur on a time scale of tens of picoseconds).(82) Dipole-relaxational motions on the nanosecond time scale have been observed for a variety of proteins. For example, such motions were recorded for apohemoglobin and bovine serum albumin0 04 105) labeled with the fluorescent probe 2,6-TNS. 

Femtosecond spectroscopy has an ideal temporal resolution for the study of ultrafast water motions from femtosecond to picosecond time scales [33-36]. Femtosecond solvation dynamics is sensitive to both time and length scales and can be a good probe for protein hydration dynamics [16, 37-50]. Recent femtosecond studies by an extrinsic labeling of a protein with a dye molecule showed certain ultrafast water motions [37-42]. This kind of labeling usually relies on hydrophobic interactions, and the probe is typically located in the hydrophobic crevice. The resulting dynamics mostly reflects bound water behavior. The recent success of incorporating a synthetic fluorescent amino acid into the protein showed another way to probe protein electrostatic interactions [43, 48]. 

The studies of water motions around proteins have been difficult because of the lack of a reliable optical probe. Early attempts to use an extrinsic labeling of a protein with a dye molecule showed certain mobile water molecules [37-42], Such extrinsic labeling has limited probing sites and thus prevents general applications. We proposed to use the intrinsic tryptophan residue as a local... 

This simple example shows that even though only 26% of the protein molecules contain more than one chelating group, 63% of the signal will come from these multiply labeled molecules Refinements in the mathematical model will not change this conclusion qualitatively. Since the more chelators the product contains, the less it resembles the unmodified protein, it is evident that with a nonselective reagent the average number of chelators per protein must be kept well below 1.0 if the labeled product is to retain its native properties. 

The aim of this study was to show wide spectrum of possible labels for proteins and the techniques for incorporating them into protein molecules. The choice of label used and the method of labeling is closely related to the purpose for which the labeled protein is to be employed. Although there are many different approaches, they can often fulfil the same final goal (e.g. various protein labels in immunoanalysis). 

Single-molecule spFRET fluorescence trajectories contain detailed information about the conformational motion associated with the enzymatic turnovers. The upper panel in Fig. 24.4 shows an expanded portion of a trajectory (middle panel) recorded from donor fluorescence of a single-pair donor-acceptor labeled protein with substrate present. By comparison, the lower panel shows a portion of a donor-fluorescence trajectory recorded from a donor-only labeled T4 lysozyme under the same conditions. The... 

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