HLP: A webserver for predicting half-life of peptides in intestine like environment
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Predict peptide half-life and antibacterial activity in batch mode.
Simultaneously, a number of peptides can be submitted to predict their half-life and antibacterial activity.


HLP is a web server developed for predicting half-life of peptides in intestine like environment. In order to increase/decrease half-life, it allows users to generate mutants for their query peptide sequence and predicts the half-life accompanied with physicochemical properties for original as well as mutant peptides. Also, it may scan user submitted protein sequence in overlapping window and helps retrieving peptides having high half-life. Batch submission is another intresting feature of HLP that helps in high throughput screening of peptides with high half-life. This page is designed for providing help on HLP. Please click on topic or subtopic for detail help.

Help TopicDescription
Input SequenceInput peptide/protein sequence(s) to be used on web server for Peptide, Protein and Batch modules.
Criteria for Model SelectionDescribes model to be used depending upon the length of query peptide(s) sequence(s).
Peptide (Help)   
Use Module
This module predicts half-life and calculate physicochemical properties for user submitted original peptide and its all possible mutants.
Protein (Help)   
Use Module
Generate peptides (in overlapping window) from user submitted protein sequence to predict half-life and calculate physicochemical properties for them.
Batch (Help)   
Use Module
Helps in high throughput screening of peptides to prioritize high half-life and better physicochemical properties possessing peptides from large number of peptides.
Scanning of ProteinDescribes the procedure to scan a protein to generate peptides from it.
Result TableDetailed description of table containing results.

Input Sequence:

Input Sequence for Peptide/Protein Module(s): Peptide/Protein sequence(s) should be pasted directly (in the relevant text input box provided) in single letter code of amino acids (in single line only). Any unusual character like new line, > sign etc. can lead to an error.
Input Sequence for Batch Module: This module accepts the peptide sequences only. The input peptides should be pasted in textbox / uploaded through a text file in FASTA format only.

Note: Example input sequences (in form of a button named "Use Example Sequence(s)") are given with each module.
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Criteria for Model Selection on Peptide, Protein and Batch modules

We have developed a total of four models to use on our HLP web server. Out of these four, two models have been developed using Support Vector Machine (SVM) and make the use of amino acid composition, dipeptide composition for 16mer, 10mer peptide half-life prediction, respectively. Another two models have been developed using WEKA and make the use of dipeptide composition for selected dipeptides to predict the half life of 10mer and 16mer peptides. It is the user's choice for what length of peptide he/she want to predict half-life, but the selection of model should be in accordance with peptide length (i.e., if selected model is 10mer, the peptide should be minimum 10 residues long or if the selected model is 16mer, submitted peptide should be minimum 16 residues long). The "User-selected sequence length" model is just an additional feature of HLP (not actually a model) that can be used for designing of peptides 5-30 residues long. If user submits a protein/peptide more than 30 residue long, first 30 residues will be used by the Web server/software. Before using "User-selected sequence length" model, please read the flow chart given below:

Figure 1: Flow chart showing criteria for model selection.

Selection of Model (for Half-life Prediction) for User Selected Peptide Length:
Note: This is extrapolation of our models; we cannot validate our models of on variables length of peptides as we have only 10mers and 16mers.
(a) If submitted peptide is less than 16 residue long, then half-life is predicted by 10mer model.
(b) If submitted peptide is 16 residue or longer, half-life is predicted by 16mer model.

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Peptide Module:

This module permits users to paste their peptide sequences (10mer / 16mer / 5 to 30 residue long) in single letter code of amino acids to design mutants and predict half-life for original and mutant peptides. Additionally, some important physicochemical properties such as hydrophobicity, pKa, pKb, Residue volume, etc. too calculated for original as well as mutant peptides. To optimize the half-life and physicochemical properties, all the mutants may be further mutated through single mouse click over them. The entire procedure can be described through the diagram given below:

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Protein Module:

This module may be used to scan a protein sequence (of any length, needs to be single lined) to retrieve overlapping peptides (with their predicted half-life and calculated physicochemical properties) from it. Therefore, the module helps in the identification of peptides (with desired half-life and physicochemical properties) from a protein. The entire procedure is described in the diagram given below:

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Scanning of Protein Throughout its Length (To generate overlapping peptides)

The user submitted protein is scanned throughout its length (in the form of overlapping window) and all possible peptide fragments are generated. Half-life (sec) and physicochemical properties are calculated for these peptides. Thus whole protein is scanned throughout its length and stable peptides are obtained. The figure to show scheme of peptide generation is given below:

Figure 2: Diagram showing generation of overlapping peptides (10mer) from a user submitted protein sequence

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Batch Module:

Batch module has been specifically designed for high-throughput screening of peptide sequences and thus helps in prediction of half-life for multiple peptide sequences in one turn rather than submitting again and again through Peptide module. Output results may be displayed in two modes viz; Interactive / Non-interactive mode. Interactive mode allows users to sort their results values in ascending as well as descending orders. Mutation of user submitted peptides is possile just through single mouse click. In contrast to this, non-interactive mode permits to download the results in text format (tab-delimited). These results can be further used to filter various selected values. The usage of Batch module is represented in the diagram given below:

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Result Table

Results of your query are shown on new page. At this, second column named Peptides is clickable and single click action will generate mutants of that particular peptide with desired properties. First row (with S.No. "0") is always the user input prediction.

Description of fields in result table

  1. Sr.No.:    It contains track of peptides.

  2. Peptides:   This column contains original peptide submitted by any user as well as mutant peptides generated from that. Red colored residue is mutated residue.

  3. Mutation Position:   Gives information about position where mutation has been done in original peptide.

  4. Half-life (sec):    Contains half-life for original and mutated peptides. The half-life is calculated in seconds and determines stability of peptide, greater the half-life more will be stability of peptide sequence and vice-versa. The dataset for model developed for half-life prediction was taken from Gorris et. al.(2009) Rapid profiling of peptide stability in proteolytic environments.

  5. Stability:    The following conditions were used to definne stability of peptides:
    If half-life (sec) < 0.1 then Stability = Low
    Half-life (sec) 0.1 to 1.0, Stability = Normal
    Half-life (sec) > 1.0, Stability = High

  6. HPLC parameter:    They are a set of hydrophilicity high-performance liquid chromatography (HPLC) parameters (derived from the retention times of 20 model synthetic peptides, Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X was substituted with the 20 amino acids found in proteins) and correlated best with antigenicity.

    HPLC parameter (Parker et al., 1986)

  7. Hydrophobicity (KJ/mol):   Hydrophobicity index is a measure of the relative hydrophobicity, or how soluble an amino acid is in water. In a protein, hydrophobic amino acids are likely to be found in the interior, whereas hydrophilic amino acids are likely to be in contact with the aqueous environment Prabhakaran, M. 1990 Biochem. J. 269, 691-696.

  8. pKa:   pKa for a given peptide sequence is the sum of negative of the logarithm of the dissociation constant for the -COOH group values given by (D R Lide Hand Book of Chemistry and Physics, 72nd Edition, CRC Press, Boca Raton, FL, 1991).

  9. pKb:   pKb for a given peptide sequence is the sum of negative of the logarithm of the dissociation constant for the α-NH3+ group values given by (D R Lide Hand Book of Chemistry and Physics, 72nd Edition, CRC Press, Boca Raton, FL, 1991).

  10. Residue volume:   It is the sum of residue volume values as calculated by (Goldsack-Chalifoux, 1973).

  11. Molecular weight:   It is molecular weight for given peptide sequence, derived by sum of the values given for individual amino acids (after removing molecular weight for water molecules that were lost during peptide bond formation) by (Fasman, 1976).

  12. Isoelectric point (pI):   pI is the pH at the isoelectric point. This value is average of pI values given for individual amino acids. (Zimmerman et al., 1968).

  13. Surface Accessibility:   The surface accessibility for a given peptide sequence is sum of average accessibility surface area values for individual amino acids given by (Janin et al., J. Mol. Biol. 125, 357 (1978).

  14. Flexibility:   It determines symmetric/asymmetric distribution of amino acid residues in the protein molecules and for a given peptide it is sum of Amino acid scale values given by (Bhaskaran and Ponnuswamy 1988)

  15. Charge:   It is overall charge calculated for a particular peptide sequence.

  16. Polarity:   Sum of polarity values (for individual amino acids) calculated by (Ponnuswamy et al., Biochim. Biophys. Acta 623, 301 (1980).

  17. Relative Mutability:   Sum of relative mutability values (for individual amino acids) by Dayhoff et al., 1978b.

  18. Free Energy of Solution in water (kcal/mole):   As given by Charton-Charton, 1982. [PMID:7183857]

  19. Optical Rotation:   Average of optical rotation values (for individual amino acids) as given by Fasman, 1976.

  20. Entropy of Formation:   Sum of entropy of formation values (given for individual amino acid) by Hutchens, 1970.

  21. Heat Capacity:   Sum of heat capacity values (given for individual amino acid) by Hutchens, 1970.

  22. Relative Stability:   The average of relative stability scale values for individual amino acids, extracted from mutation experiments (Zhou-Zhou, 2004). [PMID:14696193]

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