Protein properties
Molecular weight, pI, GRAVY, extinction coefficient and composition from a pasted sequence.
Molecular weight, pI, GRAVY, extinction coefficient and composition from a pasted sequence.
Paste one sequence, or a FASTA file of chains that belong to the same protein, and the page gives the numbers a protein is usually described by: molecular weight, isoelectric point, the GRAVY hydropathy average, the molar extinction coefficient at 280 nm, the net charge at any pH you choose, and the full amino acid composition. Everything is computed from the sequence alone, in your browser.
Each residue adds its own mass, and one water is added back for the two ends of the chain, because every peptide bond released one:
The residue masses are the standard average masses (Gly 57.0519, Ala 71.0788, Trp 186.2132 and so on, from the amino acid table). A monoisotopic mass, the one a high-resolution mass spectrometer measures, is given under Details; it uses the mass of the most common isotope of each atom and is about 9 Da lower for a 14 kDa protein. Neither number includes disulfide bonds, which remove 2.016 Da each, or any modification: a phosphate adds 79.98 Da, and N-terminal Met removal takes 131.2 Da off.
The pI is the pH at which the net charge is zero. The net charge at a given pH is the sum over every ionisable group, each one titrating according to Henderson-Hasselbalch:
The basic groups are the N-terminus, Lys, Arg and His; the acidic ones are the C-terminus, Asp, Glu, Cys and Tyr. The pKa values are the Bjellqvist set used by Expasy ProtParam: Asp 4.05, Glu 4.45, Cys 9.0, Tyr 10.0, His 5.98, Lys 10.0, Arg 12.0, C-terminus 3.55, N-terminus 7.5, with residue-specific values for the two termini. The pH that makes q zero is found by bisection between 0 and 14. Every chain in the input contributes its own termini, so a two-chain protein is titrated as one molecule. For a single buffer component instead, use theHenderson-Hasselbalch calculator.
Absorbance at 280 nm comes almost entirely from tryptophan and tyrosine, with a small contribution from disulfide-bonded cysteine pairs. The coefficients are those of Pace and colleagues (1995), measured in water:
Two values are worth having. The reduced one assumes free cysteines and is the default here; the cystine one assumes every pair of cysteines forms a disulfide, and is under Details. A protein with no Trp and no Tyr has essentially no 280 nm signal and must be quantified another way. To turn an A280 reading into a concentration with this coefficient, use the A280 protein concentration calculator, and for the general Beer-Lambert form the Beer-Lambert calculator.
The grand average of hydropathy is the mean Kyte-Doolittle value over the chain, running from Ile at +4.5 to Arg at −4.5. Positive means hydrophobic overall, negative hydrophilic. It is a single number for the whole sequence, so it is useful for comparing constructs and for spotting a membrane protein, not for locating a transmembrane helix.
"Try an example" loads hen egg white lysozyme, the mature 129 residue chain. The tool gives a molecular weight of 14,313 Da, a pI of 9.32, a GRAVY of −0.472 and a reduced ε₂₈₀ of 37,470 M⁻¹ cm⁻¹ from its 6 Trp and 3 Tyr (6 × 5500 + 3 × 1490). The 8 cysteines form 4 disulfides in the folded protein, which raises ε₂₈₀ to 37,970 M⁻¹ cm⁻¹, so a 1 g/L solution reads A280 = 2.618 with free cysteines and 2.653 with the disulfides formed. Lysozyme is a basic protein: 17 Lys and Arg against 9 Asp and Glu leave a net charge of +7.8 at pH 7, which is why it binds cation exchangers at neutral pH. The monoisotopic mass, under Details, is 14,303.88 Da.
The net charge box inside the result takes any pH between 0 and 14. Below the pI the protein is positive, above it negative, and a protein is least soluble near its pI, where the net charge is zero. For ion exchange, pick a buffer pH about one unit away from the pI: below it for a cation exchanger, above it for an anion exchanger. The charge curve under Details shows the whole titration, with the pI marked.
For the mass of a given number of moles, or the reverse, use theprotein weight and molarity converter, and for assay standards the protein concentration calculator. To get a protein sequence from DNA first, use thetranslation tool.
The Bjellqvist set, the one behind Expasy ProtParam: Asp 4.05, Glu 4.45, Cys 9.0, Tyr 10.0, His 5.98, Lys 10.0, Arg 12.0, with a C-terminus at 3.55 and an N-terminus at 7.5. The two termini also have residue-specific values, so a chain starting in Pro is given 8.36 and one ending in Glu is given 4.75. Hen egg lysozyme comes out at pI 9.32 here, the same value ProtParam reports.
A calculated pI is a sum of textbook pKa values for the free side chains. In a folded protein the local environment shifts those pKa values, buried groups may not titrate at all, and phosphorylation, glycosylation or a His tag change the balance. Gels and isoelectric focusing therefore often land a few tenths of a unit away, sometimes a whole unit for a protein with many buried charges.
Use the reduced value for a protein in a reducing buffer, or one whose cysteines are free. Use the cystine value when the disulfides are formed, which is normal for a secreted or extracellular protein. The difference is 125 M⁻¹ cm⁻¹ per disulfide, so it only matters for proteins with several bonds and few tryptophans.
Most soluble globular proteins fall between −0.5 and 0.0. Values above about 0.4 point to a membrane protein or a very hydrophobic peptide, and strongly negative values below −1 are usual for highly charged or disordered proteins. GRAVY is an average over the whole chain, so it says nothing about where the hydrophobic stretches are.
Yes. A multi-record FASTA is treated as one protein made of those chains, which is what you want for a heterodimer or an antibody: the masses and extinction coefficients add, and every chain contributes its own pair of termini to the charge. Per-chain values are listed under Details.