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This technique can also be used to determine whether the [[Proteinogenic_amino_acid#Side_chain_properties|side chain]] of a specific [[amino acid|residue]] plays a significant role in [[bioactivity]]. This is usually accomplished by [[site-directed mutagenesis]] or randomly by creating a [[PCR]] [[High-throughput screening|library]]. Furthermore, computational methods to estimate thermodynamic parameters based on a theoretical alanine substitutions have been developed.
This technique can also be used to determine whether the [[Proteinogenic_amino_acid#Side_chain_properties|side chain]] of a specific [[amino acid|residue]] plays a significant role in [[bioactivity]]. This is usually accomplished by [[site-directed mutagenesis]] or randomly by creating a [[PCR]] [[High-throughput screening|library]]. Furthermore, computational methods to estimate thermodynamic parameters based on a theoretical alanine substitutions have been developed.


This technique is rapid, because many side chains are analyzed simultaneously and the need for protein purification and biophysical analysis is circumvented.<ref name=Liaomoqi>Weiss GA, Watanabe CK, Zhong A, Goddard A, and Sidhu SS. Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc. Natl. Acad. Sci. U S A. Aug 2000 1; 97(16): 8950-4</ref> It is a mature technology which has been using for a long period of time. It is also very utility and widely used in biology, chemistry and biochemistry field. The data can be tested by [[IR]], [[NMR Spectroscopy]], mathematical method and bioassay etc.<ref>Simonsen, S. M.; Sando, L.; Rosengren, K. J.; Wang, C. K.; Colgrave, M. L.; Daly, N. L.; Craik, D. J., Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity. Journal of Biological Chemistry 2008, 283 (15).</ref><ref> Howlader, M. T. H.; Kagawa, Y.; Miyakawa, A.; Yamamoto, A.; Taniguchi, T.; Hayakawa, T.; Sakai, H., Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa. Applied and Environmental Microbiology 2010, 76 (3).</ref><ref>Gauguin, L.; Delaine, C.; Alvino, C. L.; McNeil, K. A.; Wallace, J. C.; Forbes, B. E.; De Meyts, P., Alanine scanning of a putative receptor binding surface of insulin-like growth factor-I. Journal of Biological Chemistry 2008, 283 (30).</ref>
This technique is rapid, because many side chains are analyzed simultaneously and the need for protein purification and biophysical analysis is circumvented.<ref name="Weiss_2000">{{cite journal | author = Weiss GA, Watanabe CK, Zhong A, Goddard A, Sidhu SS | title = Rapid mapping of protein functional epitopes by combinatorial alanine scanning | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 97 | issue = 16 | pages = 8950–4 | year = 2000 | month = August | pmid = 10908667 | pmc = 16802 | doi = 10.1073/pnas.160252097 }}</ref> It is a mature technology which has been using for a long period of time. It is also very utility and widely used in biology, chemistry and biochemistry field. The data can be tested by [[IR]], [[NMR Spectroscopy]], mathematical method and bioassay etc.<ref name="Simonsen_2008">{{cite journal | author = Simonsen SM, Sando L, Rosengren KJ, Wang CK, Colgrave ML, Daly NL, Craik DJ | title = Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity | journal = J. Biol. Chem. | volume = 283 | issue = 15 | pages = 9805–13 | year = 2008 | month = April | pmid = 18258598 | doi = 10.1074/jbc.M709303200 }}</ref><ref name="Howlader_2010">{{cite journal | author = Howlader MT, Kagawa Y, Miyakawa A, Yamamoto A, Taniguchi T, Hayakawa T, Sakai H | title = Alanine scanning analyses of the three major loops in domain II of Bacillus thuringiensis mosquitocidal toxin Cry4Aa | journal = Appl. Environ. Microbiol. | volume = 76 | issue = 3 | pages = 860–5 | year = 2010 | month = February | pmid = 19948851 | pmc = 2813026 | doi = 10.1128/AEM.02175-09 }}</ref><ref name="pmid18502759">{{cite journal | author = Gauguin L, Delaine C, Alvino CL, McNeil KA, Wallace JC, Forbes BE, De Meyts P | title = Alanine scanning of a putative receptor binding surface of insulin-like growth factor-I | journal = J. Biol. Chem. | volume = 283 | issue = 30 | pages = 20821–9 | year = 2008 | month = July | pmid = 18502759 | pmc = 3258947 | doi = 10.1074/jbc.M802620200 }}</ref>


One good example of alanine scanning is the examination of the role of charged residues on the surface of proteins.<ref name="pmid21209000">{{cite journal | author = Edelheit O, Hanukoglu I, Dascal N, Hanukoglu A | title = Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis | journal = Am. J. Physiol. Renal Physiol. | volume = 300 | issue = 4 | pages = F887–97 | year = 2011 | month = April | pmid = 21209000 | doi = 10.1152/ajprenal.00648.2010 }}</ref> In a systematic study on the roles of conserved charged residues on the surface of epithelial sodium channel ([[ENaC]]), alanine scanning was used to reveal the importance of charged residues for the process of transport of the proteins to the cell surface.<ref name="pmid21209000" />
One good example of alanine scanning is the examination of the role of charged residues on the surface of proteins.<ref name="pmid21209000">{{cite journal | author = Edelheit O, Hanukoglu I, Dascal N, Hanukoglu A | title = Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis | journal = Am. J. Physiol. Renal Physiol. | volume = 300 | issue = 4 | pages = F887–97 | year = 2011 | month = April | pmid = 21209000 | doi = 10.1152/ajprenal.00648.2010 }}</ref> In a systematic study on the roles of conserved charged residues on the surface of epithelial sodium channel ([[ENaC]]), alanine scanning was used to reveal the importance of charged residues for the process of transport of the proteins to the cell surface.<ref name="pmid21209000" />
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== Applications of Alanine Scanning ==
== Applications of Alanine Scanning ==


Alanine Scanning was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor.<ref>Weiss GA, Watanabe CK, Zhong A, Goddard A, and Sidhu SS. Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc. Natl. Acad. Sci. U S A. Aug 2000 1; 97(16): 8950-4</ref> Each amino acids in the side chains was substituted by alanine. Then shotgun scanning method which combines the concepts of alanine scanning mutagenesis and binomial mutagenesis with phage display technology was used.
Alanine Scanning was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor.<ref name="Weiss_2000"/> Each amino acids in the side chains was substituted by alanine. Then shotgun scanning method which combines the concepts of alanine scanning mutagenesis and binomial mutagenesis with phage display technology was used.


Another critical application of alanine scanning is to determine the influence of individual residues on structure and activity in the prototypic [[cyclotides]] kalata B1.<ref>Simonsen, S. M.; Sando, L.; Rosengren, K. J.; Wang, C. K.; Colgrave, M. L.; Daly, N. L.; Craik, D. J., Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity. Journal of Biological Chemistry 2008, 283 (15).</ref> Cyclotides display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. On the structure of cyclotides kalata B1, all 23 non-cysteine residues were successively substituted with alanine. The data were tested by [[NMR Spectroscopy]].
Another critical application of alanine scanning is to determine the influence of individual residues on structure and activity in the prototypic [[cyclotides]] kalata B1.<ref name="Simonsen_2008"/> Cyclotides display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. On the structure of cyclotides kalata B1, all 23 non-cysteine residues were successively substituted with alanine. The data were tested by [[NMR Spectroscopy]].


In addition, alanine scanning is also used to determine which functional motif of Cry4Aa has the mosquitocidal activity.<ref> Howlader, M. T. H.; Kagawa, Y.; Miyakawa, A.; Yamamoto, A.; Taniguchi, T.; Hayakawa, T.; Sakai, H., Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa. Applied and Environmental Microbiology 2010, 76 (3).</ref> Cry4Aa was produced by [[Bacillus thuringiensis]]. It is a dipteran-specific toxin and it plays an important role in how to produce a bioinsecticide to control mosquitoes. So, it is very essential to determine which functional motif of Cry4Aa contributes to this activity. In this study, several Cry4Aa mutants were made by replacing the residues of potential receptor binding site, loops 1, 2, and 3 in domain II with alanine. A bioassay [[Culex pipiens]] was followed to test the activities.
In addition, alanine scanning is also used to determine which functional motif of Cry4Aa has the mosquitocidal activity.<ref name="Howlader_2010"/> Cry4Aa was produced by [[Bacillus thuringiensis]]. It is a dipteran-specific toxin and it plays an important role in how to produce a bioinsecticide to control mosquitoes. So, it is very essential to determine which functional motif of Cry4Aa contributes to this activity. In this study, several Cry4Aa mutants were made by replacing the residues of potential receptor binding site, loops 1, 2, and 3 in domain II with alanine. A bioassay [[Culex pipiens]] was followed to test the activities.


== References ==
== References ==

Revision as of 05:36, 12 October 2012

In molecular biology, alanine scanning is a technique used to determine the contribution of a specific residue to the stability or function of given protein.[1] Alanine is used because of its non-bulky, chemically inert, methyl functional group that nevertheless mimics the secondary structure preferences that many of the other amino acids possess. Sometimes bulky amino acids such as valine or leucine are used in cases where conservation of the size of mutated residues is needed.

This technique can also be used to determine whether the side chain of a specific residue plays a significant role in bioactivity. This is usually accomplished by site-directed mutagenesis or randomly by creating a PCR library. Furthermore, computational methods to estimate thermodynamic parameters based on a theoretical alanine substitutions have been developed.

This technique is rapid, because many side chains are analyzed simultaneously and the need for protein purification and biophysical analysis is circumvented.[2] It is a mature technology which has been using for a long period of time. It is also very utility and widely used in biology, chemistry and biochemistry field. The data can be tested by IR, NMR Spectroscopy, mathematical method and bioassay etc.[3][4][5]

One good example of alanine scanning is the examination of the role of charged residues on the surface of proteins.[6] In a systematic study on the roles of conserved charged residues on the surface of epithelial sodium channel (ENaC), alanine scanning was used to reveal the importance of charged residues for the process of transport of the proteins to the cell surface.[6]

See also

Applications of Alanine Scanning

Alanine Scanning was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor.[2] Each amino acids in the side chains was substituted by alanine. Then shotgun scanning method which combines the concepts of alanine scanning mutagenesis and binomial mutagenesis with phage display technology was used.

Another critical application of alanine scanning is to determine the influence of individual residues on structure and activity in the prototypic cyclotides kalata B1.[3] Cyclotides display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. On the structure of cyclotides kalata B1, all 23 non-cysteine residues were successively substituted with alanine. The data were tested by NMR Spectroscopy.

In addition, alanine scanning is also used to determine which functional motif of Cry4Aa has the mosquitocidal activity.[4] Cry4Aa was produced by Bacillus thuringiensis. It is a dipteran-specific toxin and it plays an important role in how to produce a bioinsecticide to control mosquitoes. So, it is very essential to determine which functional motif of Cry4Aa contributes to this activity. In this study, several Cry4Aa mutants were made by replacing the residues of potential receptor binding site, loops 1, 2, and 3 in domain II with alanine. A bioassay Culex pipiens was followed to test the activities.

References

  1. ^ Morrison KL, Weiss GA (2001). "Combinatorial alanine-scanning". Curr Opin Chem Biol. 5 (3): 302–7. doi:10.1016/S1367-5931(00)00206-4. PMID 11479122. {{cite journal}}: Unknown parameter |month= ignored (help)
  2. ^ a b Weiss GA, Watanabe CK, Zhong A, Goddard A, Sidhu SS (2000). "Rapid mapping of protein functional epitopes by combinatorial alanine scanning". Proc. Natl. Acad. Sci. U.S.A. 97 (16): 8950–4. doi:10.1073/pnas.160252097. PMC 16802. PMID 10908667. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  3. ^ a b Simonsen SM, Sando L, Rosengren KJ, Wang CK, Colgrave ML, Daly NL, Craik DJ (2008). "Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity". J. Biol. Chem. 283 (15): 9805–13. doi:10.1074/jbc.M709303200. PMID 18258598. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  4. ^ a b Howlader MT, Kagawa Y, Miyakawa A, Yamamoto A, Taniguchi T, Hayakawa T, Sakai H (2010). "Alanine scanning analyses of the three major loops in domain II of Bacillus thuringiensis mosquitocidal toxin Cry4Aa". Appl. Environ. Microbiol. 76 (3): 860–5. doi:10.1128/AEM.02175-09. PMC 2813026. PMID 19948851. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  5. ^ Gauguin L, Delaine C, Alvino CL, McNeil KA, Wallace JC, Forbes BE, De Meyts P (2008). "Alanine scanning of a putative receptor binding surface of insulin-like growth factor-I". J. Biol. Chem. 283 (30): 20821–9. doi:10.1074/jbc.M802620200. PMC 3258947. PMID 18502759. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  6. ^ a b Edelheit O, Hanukoglu I, Dascal N, Hanukoglu A (2011). "Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis". Am. J. Physiol. Renal Physiol. 300 (4): F887–97. doi:10.1152/ajprenal.00648.2010. PMID 21209000. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

Further reading

  • Michels CA (2002). Genetic techniques for biological research: a case study approach. London: J. Wiley. p. 111. ISBN 0-471-89921-6.
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