Protein Properties: What Sequence-Based Results Mean
A protein sequence can provide useful theoretical properties before any experimental measurement is available. This guide explains the results in the Protein Properties Calculator, including molecular weight, pI, charge, amino acid percentages, aromaticity, GRAVY, and extinction coefficients.
The quick reference
| Property | What it describes | Common use |
|---|---|---|
| Length | Number of residues in the entered sequence | Construct and protein-size checks |
| Molecular weight | Theoretical mass of the unmodified chain | SDS-PAGE, mass and mole calculations |
| pI and charge | Sequence-based ionization estimate | Electrophoresis and purification planning |
| Composition | Count and percentage of every amino acid | Sequence comparison and residue auditing |
| Aromaticity | Fraction of Phe, Trp, and Tyr | Sequence characterization |
| GRAVY | Mean Kyte–Doolittle hydropathy | Overall hydrophobicity comparison |
| A280 extinction | Theoretical absorbance contribution from Trp, Tyr, and cystine | Protein concentration by UV absorbance |
Amino acid counts and percentages
The composition table counts each residue and divides that count by the complete sequence length. For an amino acid i:
percentage(i) = count(i) ÷ total residues × 100
For example, 34 alanines in a 341-residue protein correspond to 34 ÷ 341 × 100 = 9.97%, displayed as 10.0% when rounded to one decimal place. Percentages use residue count, not amino acid mass.
All 20 standard amino acids remain visible even when their count is zero. This makes reports consistent and helps when comparing different sequences.
Average and monoisotopic molecular weight
The calculator adds the free-amino-acid masses and subtracts one water molecule for every peptide bond:
MW = Σ(amino acid masses) − (number of residues − 1) × mass of H₂O
The average molecular weight uses the natural average of stable isotopes and is generally the most practical value for routine lab calculations. The monoisotopic molecular weight uses the exact mass of the most abundant isotope of each element and is useful when comparing sequence-derived masses with high-resolution mass spectrometry.
Both values describe the entered, unmodified linear chain. They do not automatically add or remove signal peptides, initiator methionine, affinity tags, glycans, phosphorylation, acetylation, bound metals, cofactors, or other modifications. Disulfide formation also changes intact mass slightly, but the displayed MW does not assume a disulfide pattern.
Theoretical pI, net charge, and the charge curve
Net charge is estimated from the N-terminus, C-terminus, and ionizable side chains at the selected pH. The theoretical isoelectric point is the pH where that calculated net charge crosses zero.
Bjellqvist, Lehninger, and EMBOSS use different pKa assumptions, so their results can differ even for the same sequence. The dedicated Protein pI and pKa Scale Guide lists every value, explains the terminal-residue rules, and shows when the choice matters most.
Aromaticity
Aromaticity is the fraction of phenylalanine (F), tryptophan (W), and tyrosine (Y) residues in the sequence:
aromaticity = (F + W + Y) ÷ total residues
A result of 0.079 means that 7.9% of the sequence consists of these three aromatic amino acids. This is a composition statistic, not a direct measurement of folding, absorbance, or stability.
GRAVY: Grand Average of Hydropathy
GRAVY is the arithmetic mean of the Kyte–Doolittle hydropathy values for all residues:
GRAVY = Σ(Kyte–Doolittle value for each residue) ÷ total residues
- A more positive value indicates a more hydrophobic sequence overall.
- A more negative value indicates a more hydrophilic sequence overall.
- A value close to zero indicates a near-balanced whole-sequence average.
GRAVY should not be treated as a direct solubility prediction. A single average can hide local hydrophobic segments, transmembrane helices, folding, exposed surfaces, charge distribution, and the effects of buffer conditions.
A280 molar extinction coefficients
The molar extinction coefficient estimates how strongly the protein absorbs at 280 nm. The calculator reports units of M⁻¹ cm⁻¹ and follows the same sequence-count formulas used by Biopython ProtParam.
ε(reduced) = 5500 × Trp + 1490 × Tyr
ε(oxidized) = ε(reduced) + 125 × floor(Cys ÷ 2)
The reduced value assumes that cysteines are not paired as cystines. The oxidized value assumes the maximum number of cystine pairs that can be formed from the sequence. It does not predict the protein's actual disulfide-bond pattern. The theoretical value can be used with Beer–Lambert's law, A = εcl, when concentration, path length, sample purity, and experimental conditions are handled appropriately.
What O, U, B, Z, J, and X mean
| Code | Meaning | Why exact properties stop |
|---|---|---|
| O / Pyl | Pyrrolysine | Not covered by every selected property model |
| U / Sec | Selenocysteine | Not covered by every selected property model |
| B / Asx | Aspartate or asparagine | The exact residue and charge are unknown |
| Z / Glx | Glutamate or glutamine | The exact residue and charge are unknown |
| J / Xle | Leucine or isoleucine | The residues have different hydropathy values |
| X / Xaa | Unknown amino acid | Mass and other properties cannot be assigned exactly |
These codes are counted and included in percentages instead of being silently discarded. The calculator withholds exact physicochemical results when any are present because one or more models would require an unsupported or ambiguous assumption.
Worked example: C. elegans GAPDH-1 (P04970)
The built-in example uses the complete 341-residue GAPDH-1 sequence. With Bjellqvist selected and net charge requested at pH 7.0, Calcorium reports:
| Result | Calculated value |
|---|---|
| Length | 341 aa |
| Average MW | 36,382.05 Da |
| Monoisotopic MW | 36,359.67 Da |
| Theoretical pI | 7.68 |
| Net charge at pH 7.0 | +1.10 |
| Aromaticity | 0.079 |
| GRAVY | −0.022 |
| A280 ε, reduced | 32,890 M⁻¹ cm⁻¹ |
| A280 ε, oxidized | 33,015 M⁻¹ cm⁻¹ |
Copying or downloading a report
The report preview includes the selected pKa scale, calculated properties, charge table, and complete amino acid composition. The Include cleaned protein sequence checkbox is off by default. When selected, the copied and downloaded report also contains a FASTA-style header and the cleaned one-letter sequence wrapped at 60 residues per line.
Limitations of sequence-only properties
These results are theoretical descriptors of the exact sequence entered. Real proteins can differ because of processing, modifications, folding, oligomerization, ligand binding, solvent conditions, and experimental method. Confirm that the sequence represents the intended mature protein or construct, and report calculation assumptions when using a value in research records.