Benefits of Using Our Alanine Scan
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. Alanine scanning mutagenesis of protein-protein interfacial residues can be applied to a wide variety of protein complexes to understand the structural and energetic characteristics of the hot-spots.
We perform an analysis of changes in affinity, calculate the range of changes in the dissociation constant for point substitutions of amino acid residues with alanine in proteins. Thus, when ordering an alanine scan in our company, you significantly save on biological material, pay, and use of research equipment.
Predicting the effect of mutations in proteins missense mutation in structural genes may become either selectively advantageous or disadvantageous to the organism by affecting protein stability and interactions is of significant value. Here we propose a novel approach to the study of missense mutation. We showed that method can predict stability changes of a wide range of mutations occurring in the proteins.
The results of our calculations are in good agreement with experimental data.
The results obtained for proteins with a known three-dimensional structure:
1.Determination of the stability of protein complexes with point substitutions of amino acid residues taking into account the three-dimensional structure of the biocomplex, based on the introduced criterion: the condition number of pairwise potential energy of electrostatic interaction of amino acid residues.
2.The range of changes in the affinity of the dimeric complex will be obtained at each substitution. The biophysical approach developed in the article allows one to qualitatively determine key amino acid residues when interacting two polypeptide chains, as well as to estimate the range of Kd change when replacing key amino acid residues in peptides when they are bound to the target protein. Thus, we can predict the stability of protein complexes, qualitatively determine the dissociation constant, synthesize peptides with a given dissociation constant to various proteins, while allowing to increase the selectivity of peptides, increasing affinity for one protein and decreasing to others, which is an important condition for improving peptide therapy.
3. The potential energy values of the potential energy of the interaction between all amino acid residues of one protein and amino acid residues of another protein at each substitution
4. Differential Entropy Change Values for Each Change. When inserted amino acid residue in a hydrophobic pocket consisting of other amino acid residues inside the pocket with the great probability to stay amino acid residue, which has a change in differential entropy, as well as the difference between the mean square deviation (√D, where D is variance) for this replacement amino acid residue and standard deviation amino acid residue of wild type protein in a hydrophobic pocket will be minimal. It should be noted that √D correlates with lg(cond(W)), which in this formulation of the problem characterizes the stability of the biological complex.
5. The value lg(cond(W)) at each replacement. The stability criterion of the protein complex (lg(cond(W)) of pairwise electrostatic interaction of the potential energy of amino acid residues allows us to estimate the range of variation of Kd values and indicate which mutations in peptides will lead to an increase/decrease in the stability of the biological complex when they are bound to proteins. Thus, before conducting a costly, time-consuming experiment, when it is necessary to synthesize a highly selective peptide, which will be characterized by high affinity for one protein and low affinity for another protein, then the method developed by us will minimize the number of experiments to search for such a peptide by theoretical calculation of a given range for high and low Kd values.
6.Three-dimensional graph of the potential energy of electrostatic interaction between two wild-types proteins. Maps of potential energy of electrostatic interaction of amino acid residues of proteins involved, which allow visualizing the nature of the formation of a protein complex, identifying the maxima and minima of potential energy between amino acid residues of two proteins and identifying key amino acid residues that account for the maximum values of potential energy. Particular attention is paid to the nature of the interacting amino acid residues (hydrophobic, hydrophilic, charged). The analysis of interacting proteins was carried out taking into account the three-dimensional structure, where areas with the highest potential energy of electrostatic interaction were identified.
The calculation results indicate the range of changes in affinity for various substitutions of amino acid residues, but not the dissociation constant exactly
What do you need in order to perform computer alanine scanning:
- Choose a three-dimensional structure of the dimeric protein complex.
You can use the data base of this protein obtained using X-ray diffraction analysis or restore a three-dimensional structure using various servers and programs. Moreover, the results obtained will be more accurate, the more accurately the used structure in the calculations.
- Get the values of the potential energy of electrostatic interaction between all amino acid residues of one protein and all amino acid residues of another protein.
- Identify amino acid residues with a high value of the potential energy of electrostatic interaction in the maximum and minimum areas
- Replace the identified amino acid residues alternately with alanine and, for each case, calculate the interaction with another protein, and obtain the values of the potential energy of interaction.
- Write matrices for each case of alanine replacing
- Calculate differential entropy for each case
- Calculate the value of lg(cond(w))
- Perform plotting for all values and cases.