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Protein stability manual
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Protein stability manual
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Protein stability manual
1. DEVELOPED BY S. PRASANTH KUMAR Bioinformatics Laboratory, Department of Botany, University School of Sciences, Gujarat University, Ahmedabad-380 009, Gujarat, India. Website: http://prasanthvirtualbioinfolab.blogspot.com Email: firstname.lastname@example.org Ph.: +91-9909893929 © COPYRIGHTS. ALL RIGHTS RESERVED 2011.Citation: S.Prasanth Kumar and M. Meenachi. Virtual Quantification of ProteinStability using Applied Kinetic and Thermodynamic Parameters. IIOAB (In Press). THIS PROGRAM IS DISTRIBUTED UNDER CREATIVE COMMON LICENSEPERMITTING EXTENSIVE USAGE, REPRODUCTION AND DISTRIBUTION FOR ACADEMIC AND NON-ACADEMIC USERS PROVIDED THIS WORK SHOULD BE PROPERLY CITED.
Protein Stability Program- Developed by S. Prasanth Kumar PROTEIN STABILITY PROGRAM MANUAL AT A GLANCE 1. Protein Stability 1 2. Applications of Protein Stability Program 1 3. System Requirements 1 4. Downloading the Program 2 5. Input File 2 6. Running the Program 5 7. Results Interpretation 7 8. Expert Level Analysis of Developed Annex-i Algorithm ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 11. Protein StabilityProtein stability, the most important aspect of molecular dynamics andsimulations, requires sophisticated instrumentations of molecular biology toanalyze its kinetic and thermodynamic background. Protein stability isdefined by the ability of a protein to retain its structural conformation or itsactivity when subjected to physical or chemical manipulations. In otherwords, any process involved in maintaining the structure and integrity of aprotein and preventing it from degradation or aggregation. Protein stabilityis quantitatively described by the standard Gibbs energy change (∆G). Such∆G values are important properties for a quantitative comparison ofstabilities of different proteins.2. Applications of Protein Stability Program It is developed in a view that one might get a clear understanding of the protein stability from the sequence itself without the need of its 3D structure which can help us to study the protein dynamics and folding pattern which act as a prerequisite for protein characterization experiments. This program will serve as a better tool for understanding protein stability in the context of molecular dynamics and the important amino acids in the domains driving folding. Protein Stability, a first program of its kind, was developed which takes raw amino acid sequence as its input and produces energy distribution for individual amino acids and its overall stability from the applied kinetic and thermodynamic parameters.3. System Requirements Little knowledge of Terminal (for Linux Users) or Command Line Prompt (for Windows Users). MS Excel or Office Spreadsheet for executing statistics ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 24. Downloading the Program‘Protein Stability’ program can be freely downloaded from an external mirrorhttp://depositfiles.com/files/tab23x3xo . Click “FREE downloading”, then the countdown of 60 seconds will be displayed and a download button ‘Download the file’ will be displayed after the completion of countdown. After clicking the download button, it will pop up with a download “proteinstability.exe”. Select where you want to save the file (program) in your system.5. Input FileThe only input file is raw protein sequence. Here ‘raw’ means a string of 20essential amino acids without any header, comments or other features fromany sequence format. The protein of interest can be downloaded from anypublicly available databases such as NCBI, UniProtKB, PIR, etc. We willillustrate the sequence retrieval from NCBI with ‘Succinate dehydrogenase ofMycobacterium tuberculosis’ as an example. ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 3Browse the NCBI homepage in World Wide Web with the following URL:http://ncbi.nlm.nih.gov.in/Narrow done your search to ‘Protein’ with the keyword ‘Succinatedehydrogenase Mycobacterium tuberculosis’ typed in the search box and hit‘Search’ ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 4A search list will be displayed for the above keyword. Select a proteinsequence from Mycobacterium tuberculosis H374a and retrieve it by clickingits corresponding FASTA link provided below its entry.Now, copy only the raw sequence, paste in a notepad (or any text editor) andsave it in .txt format. For example, ‘sequence.txt’. It is recommended to savethe sequence file in the same location where you had previously downloadedthe program.The input file will look like this. ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 56. Running the ProgramInvoke the command line interpreter using ‘Run’ command or simply throughthe shortcut “Windows ( )’ + r. Type ‘cmd’ to open command prompt. It willlook like this (for clarity the background color and text has been changed).In order to get interpreted by command line prompt, specify the full path ofthe program with the change directory option.Change the directory to Desktop by the following commandType: cd C:UsersprashanthDesktopOpen the program by its filename and its file extensionType: proteinstability.exeNow, the program asks you to input the protein sequence file(remember that we had saved the sequence file in the desktop)Type: sequence.txt and hit “ENTER or RETURN” ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 6After hitting ENTER key, the result will be displayed. ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 77. Results InterpretationFirst, we have to find the top most 2 residues whose Gibbs free energy ofactivation is higher under kinetics column.Gibbs free energy of activation of Arginine (R) = 140.53 kcal/molGibbs free energy of activation of Leucine (L) = 130.53 kcal/molHence, this two amino acids were probably consumes the energy of activationto promote folding and contributes more for the protein hydrophobicity.Gibbs free energy of activation of Glutamate (E) = -123.97 kcal/molNoteworthy, this amino acid has negative energy as it compensates the highenergy consumed by the above 2 amino acids.Second, we have to examine the thermodynamic Gibbs energy otherwiseknowns as Gibbs free energy of denaturation corresponding to the abovementioned first 2 amino acids.Gibbs free energy of denaturation of Arginine (R) = -3207.78 kJ/molGibbs free energy of denaturation of Leucine (L) = -3143.53 kJ/molAs these amino acids reported a very low energy in terms of thermodynamics,it can promote the thermal stability of the protein in part.Gibbs free energy of denaturation of Alanine (A) = -3405.12 kJ/molFortunately, another amino acid (alanine) has an energy value near to thearginines and leucines. Hence, the alanine probably promotes stability ascompared to the rest of the amino acids.Third, we have to inspect the frequency of amino acid for the abovementioned 3 amino acids.Frequency of R in the protein = 47Frequency of L in the protein = 57Frequency of E in the protein = 49Frequency of A in the protein = 66The frequencies of all the 4 amino acids were comparatively greater. ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 8Finally, the interpretation of protein stability valuesThe descriptor ‘protein stability’ and its numerical values reflect thedistribution of hydrophobic amino acids across the protein sequence. If thehydrophobic amino acids were higher in counts, the protein stability valuewill also increase. Thus, it is highly recommended that protein stability valueand Gibbs energy in terms of hydrophobic amino acids calculated for both thekinetic and thermodynamic calculations should be analyzed and comparedsimultaneously.Kinetic calculationsProtein Stability value = 0.3963Gibbs energy in terms of hydrophobic amino acids = 528.54 kcal/molThermodynamic calculationsProtein Stability value = 0.3947Gibbs energy in terms of hydrophobic amino acids = -12881.17 kJ/molTo understand the existance of any linear relationship between thecalculated protein stability values and Gibbs free energy term forhydrophobic aminoacids from both kinetic and thermodynamic calculations,Pearson product moment correlation coefficient can be used that ranges from-1.0 to 1.0. correlationPearson product moment correlation coefficient Type of CorrelationNegative value e.g. -1.0 Negative CorrelationZero i.e 0 No CorrelationPositive value e.g. 1.0 Positive Correlation ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar 9A value of 1 was observed indicating that the protein stability value and theGibbs energy term for both the kinetic and thermodynamic calculationsproved a positive correlation. Hence, the energy term for hydrophobic aminoacids in both the calculations contributes hydrophobicity and stability and itis evident in the table below:ArginineArginine Leucine Total Gibbs energy Contribution (Arginine + in terms of (%) Leucine) hydrophobic amino acids Gibbs free energy of activation (Kinetic Calcualtions)140.53 130.53 271.06 528.54 51.28kcal/mol kcal/mol kcal/mol kcal/mol denaturation Gibbs free energy of denaturation (Thermodynamic Calculations)-3207.78 -3143.53 -6351.31 -12881.17 49.31kJ/mol kJ/mol kJ/mol kJ/molThe contribution of Arginine and Leucine was found to be 51.28 % and49.31 %, respectively.We can come to a conclusion that 1. Arginine and Leucine promotes hydrophobicity (consumes more Gibbs free energy of activation) and this more energy is compensated by a low energy consuming amino acid, Glutamate. 2. Arginine and Leucine also promotes stability as its Gibbs free energy of denaturation was very low. Comparatively, Alanine also contributes for the protein stability. 3. The frequencies of the four amino acids were greater and may play an important role in folding and stabilizing the molecule. 4. A linear relationship was observed between the estimated protein stability values and Gibbs energy term in both the calculations taken together. 5. Arginine and Leucine contributed 51.28% for the protein hydrophobicity and contributed 49.31% for the protein stability. Hence, it is evident that these two amino acids contributed half of the driving force for hydrophobicity and stabilization.From this analysis, one might get an overall idea about the importance ofamino acids in kinetics and thermodynamics point of view and the majorforce promoting the protein stability. ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar Annex-i8. Expert Level Analysis of Developed Algorithm ©Copyrights. All Rights Reserved, 2011
Protein Stability Program- Developed by S. Prasanth Kumar Annex-i ©Copyrights. All Rights Reserved, 2011
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