Protein sequencing specificity

Strip plots can only be constructed when the crosspeaks have already been assigned in the 2D HSQC spectrum. In a 15N-labeled protein, sequence-specific assignments come from sequential NOE (a,N, /3,N and N,N) crosspeaks located in the 3D HSQC-NOESY spectrum. The walk through the protein backbone is done in the same way as with unlabeled proteins, except that overlap in NOESY spectra is greatly reduced by spreading the crosspeaks out in the 15N dimension of a 3D spectrum. 

Murata, L. Dodson, M. The herpes simplex vhus type 1 origin-binding protein. Sequence-specific activation of adenosine triphosphatase activity by a double-stranded DNA containing box I.. J. Biol. Chem. 1999, 274 (52), 37,079-37,086. 

The comprehensive strategy described here aims to identify in the primary structure of voltage- and ligand-gated channel proteins sequence-specific motifs that define the pore-forming properties. Particular emphasis is on pore size and ionic selectivity of the open channel. The validity of this approach is supported by... 

Meanwhile, a combinatorial optimization method has subsequently been developed that allows a wider range of auxiliary conditions to be factored into the design of protein sequence-specific labeling protocols [109]. Overall, these tools should help to make cell-free expression and selective labeling increasingly accessible to a wider range of laboratories in membrane protein structural biology. 

The importance of linked scanning of metastable ions or of ions formed by induced decomposition is discussed in this chapter and in Chapter 34. Briefly, linked scanning provides information on which ions give which others in a normal mass spectrum. With this sort of information, it becomes possible to examine a complex mixture of substances without prior separation of its components. It is possible to look highly specifically for trace components in mixtures under circumstances in which other techniques could not succeed. Finally, it is possible to gain information on the molecular structures of unknown compounds, as in peptide and protein sequencing (see Chapter 40). 

The first six chapters of this book deal with the basic principles of protein structure as we understand them today, and examples of the different major classes of protein structures are presented. Chapter 7 contains a brief discussion on DNA structures with emphasis on recognition by proteins of specific nucleotide sequences. The remaining chapters illustrate how during evolution different structural solutions have been selected to fulfill particular functions. 

Bacteriophage repressor proteins provide excellent examples of sequence-specific interactions between the side chains of a protein and bases lining the floor of the major groove of B-DNA. As we shall see, to fit the protein s recognition module into this groove it has to be made even wider in other words, the B-DNA has to be distorted. 

Seeman, N.C., Rosenberg, J.M., Rich, A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc. Natl. Acad. Sci. USA 73 804-809, 1976. 

Sequence-specific protein-DNA interactions recognize operator regions... 

Figure 8.15 Sequence-specific protein-DNA interactions provide a general recognition signal for operator regions in 434 bacteriophage, (a) In this complex between 434 repressor fragment and a synthetic DNA there are two glutamine residues (28 and 29) at the beginning of the recognition helix in the helix-turn-helix motif that provide such interactions with the first three base pairs of the operator region.

Steitz, T.A. Stmctural studies of protein-nucleic acid interaction the sources of sequence-specific binding. 

The promoter proximal elements are usually 100 to 200 base pairs long and relatively close to the site of initiation of transcription. Within each of these elements there are DNA sequences specifically recognized by several different transcription factors which either interact directly with the preinitiation complex or indirectly through other proteins. 

Most sequence-specific regulatory proteins bind to their DNA targets by presenting an a helix or a pair of antiparallel p strands to the major groove of DNA. Recognition of the TATA box by TBP is therefore exceptional it utilizes a concave pleated sheet protein surface that interacts with the minor groove of DNA. Since the minor groove has very few sequence-specific... 

One of the most important molecular functions of p53 is therefore to act as an activator of p21 transcription. The wild-type protein binds to specific DNA sequences, whereas tumor-derived p53 mutants are defective in sequence-specific DNA binding and consequently cannot activate the transcription of p5 3-con trolled genes. As we will see more than half of the over one thousand different mutations found in p53 involve amino acids which are directly or indirectly associated with DNA binding. 

Figure 9.19 shows the sequence of the DNA that was used for the structure determination of the p53-DNA complex the bases involved in sequence-specific binding to the protein are shaded. One molecule of the DNA-bind-ing domain of p53 binds to the minor and the major grooves of the DNA making sequence-specific interactions with both strands (Figure 9.20). 

Interactions that are not sequence specific are also an Important part of the binding and occur between the sugar and phosphate residues of the DNA and the side-chain and main-chain atoms of the protein. In the crystals the DNA fragment retains the B-DNA structure with only minor distortions. 

Figure 10.6 One sequence-specific interaction occurs more frequently than others in protein-DNA complexes two hydrogen bonds form between an arginine side chain of the protein and a guanine base of the DNA, as shown in this diagram.

Proteins are usually separated into two distinct functional classes passive structural materials, which are built up from long fibers, and active components of cellular machinery in which the protein chains are arranged in small compact domains, as we have discussed in earlier chapters. In spite of their differences in structure and function, both these classes of proteins contain a helices and/or p sheets separated by regions of irregular structure. In most cases the fibrous proteins contain specific repetitive amino acid sequences that are necessary for their specific three-dimensional structure. 

Figure 16.19 Schematic drawing illustrating the structure and sequence of the RNA fragment that is recognized and bound by the coat protein of bacteriophage MS2. The RNA fragment forms a base-paired stem with a bulge at base -10 and a loop of four bases. Bases that form sequence-specific Interactions with the coat protein are red. (Adapted from a diagram provided by L. Llljas.)...

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