Interaction probe-protein

Tn general, the. solvent-accessible surface (SAS) represents a specific class of surfaces, including the Connolly surface. Specifically, the SAS stands for a quite discrete model of a surface, which is based on the work of Lee and Richards [182. They were interested in the interactions between protein and solvent molecules that determine the hydrophobicity and the folding of the proteins. In order to obtain the surface of the molecule, which the solvent can access, a probe sphere rolls over the van der Waals surface (equivalent to the Connolly surface). The trace of the center of the probe sphere determines the solvent-accessible surjace, often called the accessible swface or the Lee and Richards surface (Figure 2-120). Simultaneously, the trajectory generated between the probe and the van der Waals surface is defined as the molecular or Connolly surface. 

DNA CT also permits chemistry at a distance. Oxidative DNA damage and thymine dimer repair can proceed in a DNA-mediated reaction initiated from a remote site. These reactions too are sensitive to intervening DNA dynamical structure, and such structures can serve to modulate DNA CT chemistry. The sensitivity of DNA CT to base pair stacking also provides the basis for the design of new DNA diagnostics, tools to detect mutations in DNA and to probe protein-DNA interactions. 

The synthesis, spectral properties, and applications of symmetrical as well as unsymmetrical, hydrophobic oxo-squaraine probes for noncovalent interaction with proteins, lipids, cells, and other high-molecular-weight analytes are described in numerous publications and patents [52, 57, 58]. 

Tramier M, Gautier I, Piolot T, Ravalet S, Kemnitz K, Coppey J, Durieux C, Mignotte V, Coppey-Moisan M (2002) Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells. Biophys J 83 3570-3577... 

Massova, I. Kollman, P., Computational alanine scanning to probe protein-protein interactions a novel approach to evaluate binding free energies, J. Am. Chem. Soc. 1999,121, 8133-8143... 

The application of heterobifunctional cross-linkers allows macromolecular PAL to probe protein-protein interactions, including subunit interactions and location, monitoring the conformational changes induced by signal transmission. 

Advances in chemical synthesis have enabled considerable sophistication in the construction of diverse compound libraries to probe protein function [61, 62). However, few general techniques exist that can directly assess binding mechanisms and evaluate ligand afEnities in a multiplexed format. To realize the full potential of combinatorial chemistry in the drug discovery process, generic and efficient tools must be applied that combine mixture-based techniques to characterize protein-ligand interactions with the strengths of diversity-oriented chemical synthesis. 

There are no inherent limitations to the nature of the interaction that can be probed with the FAC method. This too stems from an uncoupling of the binding event and the detector. The method can be applied to simple binary interactions between protein and small molecule, but also to protein-protein interactions, protein-cell interactions and virtually any interaction that can be modeled in a flow system. Some of the more elegant examples include drug interaction with whole cells [12] and membrane-bound receptors from brain homogenates [13]. Ultimately, the limitations are dictated by what can be detected from a stream of column effluent. 

Maleknia, S.D. Wong, J.W.H. Downard, K.M. Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins. Photochem. Photobiol. Sci. 2004, 3, 741-748. 

Chalmers, M.J. Busby, S.A. Pascal, B.D. He, Y. Hendrickson, C.L. Marshall, A.G. Griffin, P.R. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Anal. Chem. 2006. 

Schmidt, M., Pahl, R., Ihee, H., and Srajer, V. 2005b. Protein ligand interactions probed by time-resolved X-ray structure determination. In Methods in molecular biology, vol. 305 Protein-ligand interactions Methods and Applications. G. U. Nienhaus, Ed. Totowa, NJ Humana Press. 

Frazier, R.A., Papadopoulou, A., Mueller-Harvey, I., Kissoon, D., and Green, R.J., Probing protein-tannin interactions by isothermal titration microcalorimetry, J. Agric. Food Chem., 51, 5189, 2003. 

Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (L. M. Smith et al., 19 8 5), as a label in homogeneous immunoassay systems (Nithipatikom and McGown, 1987), to investigate specific interactions of proteins with cells surfaces (Hochman et al., 1988), and as an important fluorescent tag of antibodies in immunohistochemical staining techniques (Davidson and Hilchenbach, 1990). 

A genosensor, or gene-based biosensor/DNA biosensor, normally employs immobilized DNA probes as the recognition element and measures specific binding processes such as the formation of DNA-DNA and DNA-RNA hybrids, and the interactions between proteins or ligand molecules and DNA at the sensor surface [5]. 

In Far-Western blotting the membrane is probed with another protein to detect specific protein-protein interactions (24). The reaction can be revealed using biotinylated or GST-tagged bait or probe protein followed by a streptavidin-HRP or an anti-GST-HRP chemiluminescent detection system, respectively. 

Function for Protein-Ligand Interactions Probing the Reference State. 

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]. 

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.

Maleknia, S.D. and Downard, K. (2001) Radical approaches to probe protein structure, folding, and interactions by mass spectrometry. Mass Spectrom. Rev., 20, 388—401. 

Kriwacki RW,Wu J, Siuzdak G, Wright PE (1996) Probing protein/protein interactions with mass spectrometry and isotopic labeling analysis of the p21/Cdk2 complex. J Am Chem Soc 118 5320-5321... 

Crystalline amino acids have often been used as model compounds for probing functional group interactions in proteins. The 3-site 120° jump motion of the ammonium (-NH3) group in alanine has been studied using 2H NMR lineshape analysis and by considering the anisotropy of the 2H spin-lattice relaxation [182]. The activation energy for this motion was estimated to be 40.5 kj mol-1. 

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