Alanine scan

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. 

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." ... 

Figure 3.1 Peptidomimetic chemistry attempts to produce a non-peptidic drug to mimic a bioactive peptide. In Step A, the smallest bioactive fragment of the larger peptide is identified in Step B, a process such as an alanine scan is used to identify which of the amino acids are important for bioactivity in Step C, individual amino acids have their configuration changed from the naturally occurring L-configuration to the unnatural D-configuration (in an attempt to make the peptide less naturally peptidic ) in Step D, individual amino acids are replaced with atypical unnatural amino acids and amino acid mimics in Step E the peptide is cychzed to constrain it con-formationally finally, in Step F, fragments of the cyclic peptide are replaced with bioisosteres in an attempt to make a non-peptidic organic molecule.

The first approach is shown in figure 3.1. This approach uses various techniques (e.g., alanine scanning) to identify the smallest peptide segment with biological activity within the overall peptide. This minimal bioactive segment may be cyclized or have its stereochemistry altered in order to attain restriction of conformational freedom and... 

Tang, W. J., Stanzel, M., and Gilman, A. G. (1995). Truncation and alanine-scanning mutants of type I adenylyl cyclase. Biochemistry 34, 14563-14572. 

In addition to the C-terminus, several other regions of Ga have been implicated in receptor binding by a variety of experimental approaches (Fig. 4). Residues in the a4//16 loop contribute to the binding surface as demonstrated by alanine-scanning mutagenesis (Onrust et al, 1997), studies with chimeric Ga proteins (Bae et al., 1997, 1999), sequence analysis of conserved amino acids in Ga subclasses (Lichtarge et al., 1996), chemical cross-linking (Cai et al, 2001), and protection from tryptic proteolysis (Mazzoni and Hamm, 1996). The C-terminal 11 amino acids of Gat and... 

Harlow GR, Halpert JR (1997) Alanine-scanning mutagenesis of a putative substrate recognition site in human cytochrome P4503A4. J Biol Chem 272, 5396-5402. 

Special applications of substitution analysis include the so-called amino acid walks or scans (e.g., alanine scan, glycine walk) (50,51). In this type of screening one replaces each single amino acid of the sequence with only one distinct amino acid (usually alanine or glycine). The progressive amino acid scan is achieved by a stepwise increase in the number of positions exchanged by the distinct amino acid (52). 

Clones selected under more stringent conditions showed a further improvement in binding, in combination with the best VL previously selected (Ki= 1.1 x 10"10 M). An interesting point here is that there was no correlation between the alanine scan and the variability of the positions found amongst the best binding clones (in contrast to Ref. 95). 

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