Protein-nucleic acid interactions methods

Wong, I Lohman, T.M. (1993) A Double-Filter Method for Nitrocellulose-Filter Binding. Application to Protein-Nucleic Acid Interactions, Proc. Natl. Acad. Sci. USA 90, 5428-5432. 

We focus on four interrelated aspects of protein-protein and protein-nucleic acid interactions. Section 2 deals with building structural models for protein complexes. In Section 3 we present an overview of the various methods for computing contributions to the stability of protein complexes. In Section 4 the focus shifts to the rates of forming protein complexes. Finally in Section 5 we discuss the impacts of protein dynamics on the structures, thermodynamics, and kinetics of protein complexes. 

The two-hybrid system has been used to identify interactions among the entire complement of proteins encoded by the genomes (Uetz et al, 2000). The two-hybrid system can be scaled and automated for the systematic search of protein-protein interactions. The data have proven to be valuable when integrated with protein-protein interaction data from various sources in interaction proteontics. The method can be adapted/modified to study protein-nucleic acid interactions (Putz et al, 1996 Alexander et al, 2001) and protein-ligand interactions (Licitra and Leu, 1996). 

Combined with a difference technique the method results in substantial simplifications of the complex protein nmr spectra and in an effective increase in the spectral resolution for the polarized groups. This permits detailed studies of various interactions of proteins, such as the enzyme-inhibitor interactions discussed in sections 5,2 and 6.2 for RNase A and lysozyme. For both enzymes the photo-CIDNP spectrum proved to be very sensitive to the presence of inhibitors, albeit in conpletely different ways Examples of applications to the study of protein-nucleic acid interactions will be treated by Hilbers et al. elsewhere in this volume. We believe that in spite of its brief existence the photo-CIDNP method is potentially a powerful tool for the study of protein structure in solution. 

The application of the photo-CIDNP method in conjunction with the introduction of deuterated amino acids holds great promise for the study of protein-protein and protein-nucleic acid interactions with NMR. In this contribution it has been shown that this allows partial assignment of the aromatic part of the protein spectrum in a convenient way. Binding of DNA fragments affects both the resonances of the phenylalanyl residues as well as the resonances of the tyrosines at the surface of the protein. The disappearance of the emission signals of the latter provides direct evidence for their Involvement in the interaction with DNA. 

Beside successful studies of protein-nucleic acid interactions [19, 21, 22] and protein-folding processes [23] the method has shown to be a proper tool in glyco-sciences since Tyr-, His- and Trp-residues are often constituents of the carbohydrate binding domain [5-7, 15, 24, 25]. Many plant and animal lectins harbour CIDNP-reactive amino acid residues as an essential part of their binding pocket architecture [26-28]. 

It has a potential of permitting analysis of the kinetics of protein-protein/nucleic acids/glycan/ligand interactions via real-time detection methods. 

---