Proteins surface probes

Sackett, D. L. and Wolff, J. (1987). Nile red as a polarity-sensitive fluorescent-probe of hydrophobic protein surfaces. Anal. Biochem. 167, 228-234. 

Myelin in situ has a water content of about 40%. The dry mass of both CNS and PNS myelin is characterized by a high proportion of lipid (70-85%) and, consequently, a low proportion of protein (15-30%). By comparison, most biological membranes have a higher ratio of proteins to lipids. The currently accepted view of membrane structure is that of a lipid bilayer with integral membrane proteins embedded in the bilayer and other extrinsic proteins attached to one surface or the other by weaker linkages. Proteins and lipids are asymmetrically distributed in this bilayer, with only partial asymmetry of the lipids. The proposed molecular architecture of the layered membranes of compact myelin fits such a concept (Fig. 4-11). Models of compact myelin are based on data from electron microscopy, immunostaining, X-ray diffraction, surface probes studies, structural abnormalities in mutant mice, correlations between structure and composition in various species, and predictions of protein structure from sequencing information [4]. 

The MBPs are extrinsic proteins localized exclusively at the cytoplasmic surface in the major dense line (Fig. 4-11), a conclusion based on their amino acid sequence, inaccessibility to surface probes and direct localization at the electron microscope level by immunocytochemistry. There is evidence to suggest that MBP forms dimers, and it is believed to be the principal protein stabilizing the major dense line of CNS myelin, possibly by interacting with negatively charged lipids. A severe hypomyelination and failure of compaction of the major dense line in MBP deficient shiverer mutants supports this hypothesis (Table 4-2). 

The fluorescent probe 2,6-TNS and other similar aminonaphthalene derivatives (1,8-ANS, DNS) were considered to be indicators of the polarity of protein molecules, and they were assumed to be bound only to hydrophobic sites on the protein surface. The detection of considerable spectral shifts with red-edge excitation has shown that the reason for the observed short-wavelength location of the spectra of these probes when complexed to proteins is not the hydrophobicity of their environment (or, at least, not only this) but the absence of dipole-relaxational equilibrium on the nanosecond time scale. Therefore, liquid solvents with different polarities cannot be considered to simulate the environment of fluorescent probes in proteins. 

Kawabata T, Go N (2007) Detection of pockets on protein surfaces using small and large probe spheres to find putative ligand binding sites. Bioinformatics 529 516-529... 

Kemp elimination was used as a probe of catalytic efficiency in antibodies, in non-specific catalysis by other proteins, and in catalysis by enzymes. Several simple reactions were found to be catalyzed by the serum albumins with Michaelis-Menten kinetics and could be shown to involve substrate binding and catalysis by local functional groups (Kirby, 2000). Known binding sites on the protein surface were found to be involved. In fact, formal general base catalysis seems to contribute only modestly to the efficiency of both the antibody and the non-specific albumin system, whereas antibody catalysis seems to be boosted by a non-specific medium effect. 

While the effect on tests requiring the interaction of the tumour cells with extra cellular matrix components suggest that the interaction of RAPTA compounds with cell surface molecules may be responsible for part of their activity, the compounds also accumulate within cancer cells and interact to a significant extent with proteins in the cytoplasm. Adduct formation of RAPTA compounds with proteins has been studied using mass spectrometric methods [10, 25, 26]. In general, rapid and irreversible binding has been observed. In a recent study the reactivity of cisplatin and RAPTA-C with a mixture of proteins was probed without using any... 

We recently developed a systematic method that uses the intrinsic tryptophan residue (Trp or W) as a local optical probe [49, 50]. Using site-directed mutagenesis, tryptophan can be mutated into different positions one at a time to scan protein surfaces. With femtosecond temporal and single-residue spatial resolution, the fluorescence Stokes shift of the local excited Trp can be followed in real time, and thus, the location, dynamics, and functional roles of protein-water interactions can be studied directly. With MD simulations, the solvation by water and protein (residues) is differentiated carefully to determine the hydration dynamics. Here, we focus our own work and review our recent systematic studies on hydration dynamics and protein-water fluctuations in a series of biological systems using the powerful intrinsic tryptophan as a local optical probe, and thus reveal the dynamic role of hydrating water molecules around proteins, which is a longstanding unresolved problem and a topic central to protein science. 

Figure 39. Dynamic Stokes shifts of all 16 mutants in two states. Circles and squares are the original data, and the black lines are the best fit. The names of mutants are shown on the top, and the ticks correspond to the data points. The inset in the top panel shows the physical meanings of the two Stokes shifts, A i and AE2- The insets in the lower two panels show different contributions of surface water to AEi (big arcs, light arrow) and AE2 (small ellipse, dark arrow) when tryptophan is buried (left) or exposed (right). Water molecules in the big arcs are within - 10 A around tryptophan, and water molecules in the small ellipse are those that directly interact with protein and probed by tryptophan.

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