Protein structure unknown

The basic structural unit of these two-sheet p helix structures contains 18 amino acids, three in each p strand and six in each loop. A specific amino acid sequence pattern identifies this unit namely a double repeat of a nine-residue consensus sequence Gly-Gly-X-Gly-X-Asp-X-U-X where X is any amino acid and U is large, hydrophobic and frequently leucine. The first six residues form the loop and the last three form a p strand with the side chain of U involved in the hydrophobic packing of the two p sheets. The loops are stabilized by calcium ions which bind to the Asp residue (Figure S.28). This sequence pattern can be used to search for possible two-sheet p structures in databases of amino acid sequences of proteins of unknown structure. 

A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the native protein has a molecular weight of 240,000. Chromatography in the presence of 6 M guanidine hydrochloride yields only a peak for a protein of M, 60,000. Chromatography in the presence of 6 M guanidine hydrochloride and 10 mM /3-mercaptoethanol yields peaks for proteins of M, 34,000 and 26,000. Explain what can be determined about the structure of this protein from these data. 

In general, ligand structure-based methods remain indispensable in those cases when the structure of binding site of the target protein is unknown. 

This example is one where the accurate three-dimensional structure of the protein is unknown under these circumstances, it is necessary to create a computer model. The development of inhibitors that are designed to overcome the effects of this mutation could not be based on the accurate structure of a ligand-binding site here, ligand-based design would be appropriate (see Sect. 7.9). 

The molecular replacement method assumes similarity of the unknown structure to a known one. This is the most rapid method but requires the availability of a homologous protein s structure. The method relies on the observation that proteins which are similar in their amino acid sequence (homologous) will have very similar folding of their polypeptide chains. This method also relies on the use of Patterson functions. As the number of protein structure determinations increases rapidly, the molecular replacement method becomes extremely useful for determining protein phase angles. 

Diederichs, K. Structural superposition of proteins with unknown alignment and detection of topological similarity using a six-dimensional search algorithm. Proteins Struc., Func., Genet. 1995, 23, 187-195. 

Data processing occurs in two stages. Initially, diffraction images are reduced to a tabulation of reflection indices and intensities or, after truncation, structure factors. The second stage involves conversion of the observed structure factors into an experimental electron density map. The choice of how to execute the latter step depends on the method used to determine the phase for each reflection. The software packages enumerated above generally focus on exploitation of anomalous signals to overcome the phase problem for a protein of unknown structure. 

The mechanism resembles that proposed for a phosphotriesterase (Fig. 12-24). The triesterase catalyzes detoxification of organophosphorus toxins such as parathion (Box 12-E) and seems to have evolved rapidly from a homologous protein of unknown function.721 The phosphotriesterase contains two Zn2+ ions in a dimetal center. An unusual structural feature is a carbamate group, formed from Lys 169 and C02, which provides a bridging ligand for the metal pair.721-725 A carbamylated lysine also functions in ribulose bisphos-phate carboxylase (Fig. 13-11). 

---