Alanine scanning

Scanning alanine mutagenesis investigating effects at the level of drug [209]... 

Yes. Most cytokines have a common four-helix bundle structure. Experimental techniques including scanning alanine mutagenesis have been used to show that cytokines have several regions that are recognized by their receptors. Therefore, one cytokine molecule can act to cross-link two separate receptor molecules. 

Fig. 1 Scanning curve of a chromatogram track with 100 ng per chromatogram zone of the amino acids glycine (1), alanine (2), valine (3), leucine (4).

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

Duchesnes CE, Murphy PM, Williams TJ, Pease JE. Alanine scanning mutagenesis of the chemokine receptor CCR3 reveals distinct extracellular residues involved in recognition of the eotaxin family of chemokines. Mol Immunol 2006 43(8) 1221-1231. 

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... 

Pons, J. Rajpal, A. Kirsch, J.F., Energetic analysis of an antibody/antigen interface alanine scanning mutagenesis and double mutant cycles on the HyHEL-10/lysozyme interaction, Prot. Sci. 1999, 8, 958-968. 

Massova and P. A. Kollman, Computational alanine scanning to probe protein-protein... 

Beck-Siddnger AG, Wieland HA, Wittenben H, Willim KD, Rudolf K, et al. 1994. Complete L-alanine scan of neuropeptide Y reveals ligands binding to Y1 and Y2 receptors with distinguished conformations. Eur J Biochem 225 947. 

Alternative approaches to mapping secondary structure have also been reported. These include alanine-and lysine-scanning mutagenesis, both of which do not rely on chemical modification (Yan et al., 1999 Sine et al., 2002). 

Robust peptide-derived approaches aim to identify a small drug-like molecule to mimic the peptide interactions. The primary peptide molecule is considered in these approaches as a tool compound to demonstrate that small molecules can compete with a given interaction. A variety of chemical, 3D structural and molecular modeling approaches are used to validate the essential 3D pharmacophore model which in turn is the basis for the design of the mimics. The chemical approaches include in addition to N- and C-terminal truncations a variety of positional scanning methods. Using alanine scans one can identify the key pharmacophore points D-amino-acid or proline scans allow stabilization of (i-turn structures cyclic scans bias the peptide or portions of the peptide in a particular conformation (a-helix, (i-turn and so on) other scans, like N-methyl-amino-acid scans and amide-bond-replacement (depsi-peptides) scans aim to improve the ADME properties." ... 

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