Calculation of Biochemical Parameters of Proteins with ProtParam Web Tool

ProtParam is a powerful web tool that allows the calculation of various biochemical parameters on protein structures and sequences. This tool is also used to analyze the physicochemical properties of proteins and has an important place in bioinformatics studies. ProtParam web tool is generally useful for researchers who evaluate various properties of protein sequences. ProtParam web tool can be accessed at https://web.expasy.org/protparam/ .

The Swiss-Prot/TrEMBL accession number or ID or the raw sequence (FASTA) of the protein can be specified as input to the ProtParam web tool. Gaps and numbers are ignored. If you provide an accession number for a Swiss-Prot/TrEMBL entry, you will be directed to an intermediate page that allows you to select the part of the sequence you want to analyze. This intermediate page involves selecting mature chains or peptides from the Swiss-Prot feature table. The parameters calculated by the ProtParam web tool include; molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, half-life estimate, instability index, aliphatic index and grand average of hydropathicity (GRAVY) are available (1).

Extinction Coefficient

The extinction coefficient indicates how much light a protein absorbs at a particular wavelength. Estimating this coefficient is useful for following a protein with a spectrophotometer when purifying it (1).

Half-life

Half-life is an estimate of the time it takes for half of the protein in a cell to be lost after its synthesis in the cell. This estimate is given for three organisms (human, yeast, and E. coli). ProtParam estimates half-life by looking at the N-terminal amino acid of the sequence being examined (1).

Instability Index

The instability index provides an estimate of the stability of a protein in a test tube. A protein with an instability index of less than 40 is predicted to be stable. A value above 40 predicts that the protein may be unstable (1).

Aliphatic Index

The aliphatic index of a protein is defined as the relative volume of the aliphatic side chains (alanine, valine, isoleucine, and leucine). This can be considered a positive factor for the increased thermal stability of globular proteins (1).

Grand average of hydropathicity

The grand average (GRAVY) value of hydropathicity of a peptide or protein is calculated as the sum of the hydropathicity values ​​of all amino acids and this sum is divided by the number of residues in the sequence (1).

In the sample application, the P53 protein will be used as input in ProtParam web tool.

Step 1: To obtain the UniProt ID of the P53 protein, go to the UniProt database at https://www.uniprot.org/ and search by typing the keywords “homo sapiens p53” in the search field.

Figure 1. UniProt Database Search Page.

Step 2: The human P53 protein is selected from the search page. The UniProt ID of the selected protein is seen to be P04637.

Figure 2. General Information of P53 Protein in UniProt Database.

Step 3: After obtaining the UniProt ID of the P53 protein, go to ProtParam web tool. Then, enter the ID code P04637 in the input section and click on the Compute parameters button.

Figure 3. Input Page of ProtParam Web Tool.

Step 4: In the intermediate screen before the result, the entire protein (chain), various regions and motifs are seen. From here, the "chain" part of the P53 protein is selected. The reason for this is that the aim is to examine the biochemical parameters of the entire protein.

Figure 4. Intermediate Screen of ProtParam Web Tool.

Step 5: When the results for the P53 protein are examined, the long name of the protein is shown under the Description heading and the organism it belongs to is shown under the Organism heading (Figure 4).

Figure 5. Molecular Weight, Theoretical pI and Amino Acid Composition Values ​​of P53 Protein.

When the results are examined in more detail, it is seen that the P53 protein has 393 amino acids, its molecular weight is 43653.18 Daltons and its theoretical pI value is 6.33. In addition, the percentage of each amino acid in the protein is determined (Figure 5.).

Step 6: When the results in other headings are examined, it is seen that there are 50 positively charged and 46 negatively charged amino acids in the P53 protein. The atomic composition of the P53 protein is also given in Figure 6. The extinction coefficient was obtained as 36035 at 280 nm, assuming that all cysteine ​​amino acids form cystine in water. The half-life value was estimated to be 30 hours in mammalian reticulocytes under in vitro conditions. The instability index value was observed to be 73.59, which is an indication that the P53 protein is not stable. The aliphatic index of the P53 protein was determined to be 59.08 and the grand average hydropathicity (GRAVY) value was determined to be -0.756 (Figure 6).

Figure 6. Values ​​such as Extinction Coefficient, Half-Life and Instability Index of P53 Protein.

ProtParam is a powerful tool for calculating biochemical parameters and can be used to analyze the basic properties of proteins quickly and accurately. This tool allows you to obtain important parameters such as molecular weight, isoelectric point (pI), amino acid composition by loading protein sequences. ProtParam plays an important role in both basic research and applied biotechnology studies. With its user-friendly interface and fast calculation capabilities, it is an indispensable tool for researchers working in the fields of biochemistry and bioinformatics. In addition to the physicochemical analysis of proteins, it also provides valuable information for understanding biological processes and developing new biotechnological products. ProtParam will help you save time in your scientific studies and make more in-depth analyses.

REFERENCES

1. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A.; Protein Identification and Analysis Tools on the Expasy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). pp. 571-607. https://doi.org/10.1385/1592598900