Proteins probes

For 25 years, molecular dynamics simulations of proteins have provided detailed insights into the role of dynamics in biological activity and function [1-3]. The earliest simulations of proteins probed fast vibrational dynamics on a picosecond time scale. Fifteen years later, it proved possible to simulate protein dynamics on a nanosecond time scale. At present it is possible to simulate the dynamics of a solvated protein on the microsecond time scale [4]. These gains have been made through a combination of improved computer processing (Moore s law) and clever computational algorithms [5]. 

Most of the molecules introduced in this chapter are hydrophobic. Even those molecules that have been functionalized to improve water-solubility (for example, CCVJ and CCVJ triethyleneglycol ester 43, Fig. 14) contain large hydrophobic structures. In aqueous solutions that contain proteins or other macromolecules with hydrophobic regions, molecular rotors are attracted to these pockets and bind to the proteins. Noncovalent attraction to hydrophobic pockets is associated with restricted intramolecular rotation and consequently increased quantum yield. In this respect, molecular rotors are superior protein probes, because they do not only indicate the presence of proteins (similar to antibody-conjugated fluorescent markers), but they also report a constricted environment and can therefore be used to probe protein structure and assembly. 

The techniques developed to study protein interactions can be divided into a number of major categories (Table 31.1), including bioconjugation, protein interaction mapping, affinity capture, two-hybrid techniques, protein probing, and instrumental analysis (i.e., NMR, crystallography, mass spectrometry, and surface plasmon resonance). Many of these methods are dependent on the use of an initial bioconjugation step to discern key information on protein interaction partners. 

It should also be noted that new sample probes can generate additions to the sample information queries asked of the user at the beginning of the "probe session." Thus, protein probes required the addition of queries regarding molecular weight, isoelectric point and whether biological activity is to be preserved in the chromatographic step. These questions are triggered only if the user specifies the sample as a peptide or protein in answer to the initial sample questions. Also, once the sample is specified as a protein, the question as to the pKa or pKb of the... 

Katta, V., Chait, B. T. Conformational-changes in proteins probed by hydrogen-exchange electrospray-ionization mass-spectrometry. Rapid Commun Mass Spectrom 1991, 5, 214-217. 

Table 3.2 Common Coupling Chemistries Employed for Nucleic Acid and Protein Probe Attachments... 

In this study, the effects of ionic strength and carrier protein (BSA) were examined in terms of the outcome for microarray printing. As noted previously, many factors can unduly influence printing performance. Proteins are notoriously bad when it comes to nonspecific adsorption. It should not be much of a surprise that some portion of a protein probe will adsorb to the printing device, whether it is a stainless steel quill or glass capillary. 

Isomerization process in the native and denatured photoactive yellow protein probed by subpicosecond absorption spectroscopy... 

Natural products, by virtue of their co-evolution with protein systems, possess integrated biointeractvity. The unique properties of natural products render them ideal scaffolds for activity-based protein probes. Krysiak and Breinbauer cover the use of natural products in ABPP. 

The design of most protein probes involves the incorporation of a molecular substrate as a pendant into a metal complex. The recognition of the substrate by the protein host is then revealed by changes of the photophysical properties of the probe. However, metal complexes without a specific molecular substrate have also been employed the recognition is usually on the basis of hydrophobic interactions. As can be seen in the examples mentioned in Sect. 4, the hydrophobic nature of protein matrix, in particular the substrate-binding sites, provides opportunities in the development of new probes. 

Battistuzzi G, Bellei M, Borsari M et al (2005) Axial ligation and polypeptide matrix effects on the reduction potential of heme proteins probed on their cyanide adducts. J Biol Inorg Chem 10 643-651... 

Figure 2.10. Electorphoretic mobility shift assay (EMSA). C/EBPP is a basic leucine transcription factor. An DNA oligomer consisting of the C/EBP binding site was labeled with 32P and incubated with a nuclear extract (lanes 1 and 2). The same oligomer and nuclear extract were incubated in the presence of a C/EBPP antibody (lanes 4 and 5). In lanes 1 and 2, the unbound free probe can be seen at the bottom of the autoradiogram, while probe protein complexes can be visualized in the upper section. In lanes 4 and 5 the presence of the antibody resulted in the supershift of the protein-probe complex the ternary complex consisting of the probe, C/EBPP protein, and C/EBPP antibody are now visualized at the top of the autoradiogram. The bands that were supershifted with the antibody represent C/EBPp.

Figure 21. A. Reaction of Ca2+-ATPase protein to anti-PHB IgG. Purified Ca2+-ATPase protein (8.5 pg per lane) was separated by electrophoresis on a 10% polyacrylamide gel.30 Left lane 1 Coomassie blue stain of Ca2+-ATPase protein. Left lane 2 Western blot of Ca2+-ATPase protein probed with rabbit anti-PHB IgG. Second antibody was anti-rabbit IgG conjugated to alkaline phosphatase. Color development was with the alkaline phosphatase substrate kit (Bio-Rad). B. Phosphorylation of the Ca2+-ATPase by [32PjpolyP. Purified Ca2+-ATPase protein (2 jig) was phosphorylated at room temperature by [32P]polyP and separated by electrophoresis on a 10% polyacrylamide gel.30 Right lane 1 Coomassie blue stain of phosphorylated Ca2+-ATPase. Right lane 2 Autoradiogram of phosphorylated Ca2+-ATPase.

Murphy JT, Lagarias JC. The phytofluors a new class of fluorescent protein probes. Curr. Biol. 1997 7 870-876. 57. 

Higher-Order Structure and Dynamics of FK506-Binding Protein Probed by Backbone Amide Hydrogen/Deuterium Exchange and Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry... 

V. Katta, B.T. Chait, Conformational changes in proteins probed by H/D exchange ESI-MS, Rapid Commun. Mass Spectrom., 5 (1991) 214. 

Fig. 9.7 Affinity fingerprint of a target protein probed against a chemical microarray presenting 9216 immobilized binary compounds. The color range goes from red to orange to yellow to green to blue to code for decreasing SPR signal. Rows and columns each represent one of two monomers of the binary library...

Protein-probe interactions, e.g., by DNA-binding proteins, or charge interactions between positively charged proteins (evident from binding of labeled nonspecific probes) and nucleic acid can be masked by acetylation or glycine treatments (see below). 

Of course, due to the ease of plasmid manipulation, proteins probed for interaction with the SM can be mammalian. If that is the case, it is important to understand that some post-translational modifications of mammalian proteins... 

Hart T, Hosszu LLP, Trevitt CR et al (2009) Folding kinetics of the human prion protein probed by temperature jump. Proc Natl Acad Sci USA 106 5651-5656... 

Hosszu LLP, Baxter NJ, Jackson GS, Power A, Clarke AR, Waltho JP, Craven CJ, Collinge J (1999) Structural mobility of the human prion protein probed by backbone hydrogen exchange. Nat Struct Biol 6 740... 

Barducci A, Chelli R, Procacci P, Schettino V (2005) Misfolding pathways of the prion protein probed by molecular dynamics simulations. Biophys J 88 1334... 

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