A tool to calculate protonation states for intrinsically disordered peptides and proteins

Protein Sequence
* Protein Sequence
Your protein sequence in FASTA format. Please use upper case, single-letter amino acid abbreviations. If you would like to include the terminal amino and carboxylate groups in the calculation, then add ‘n‘ before and ‘c‘ after the sequence, as follows: nGGHIFGRTc
* First Residue ID
This field allows to override the default numbering scheme of your sequence. It is useful for protein sequence fragments that do not start at residue 1
Physical Parameters
* pH start
Your titration simulation will start at the specified pH
* pH final
Your titration simulation will end at the specified pH
* Temperature [K]
Your titration simulation will be run at the specified temperature [K]. Allowed range: 273.15 - 373.15
* Ionic strength [M]
Your titration simulation will be run at the specified ionic strength [M]
* Dielectric constant

Calculate
Your titration simulation will be run assuming the specified dielectric permittivity. The default value is computed for pure water at the specified temperature, according to Uematsu, J. Phys. Chem. (1980)
* gca

* gcb
Gaussian Chain parameters defining the effective bond length ‘b' (gcb) and the shift distance ‘s' (gca). For details, see H-X Zhou, Proc Natl Acad Sci U S A. (2002) 99:3569-74.
* pKa values
Cysteine (C)
Aspartic Acid (D)
Glutamic acid (E)
Histidine (H)
Lysine (K)
Arginine (R)
Tyrosine (Y)
N-terminus (n)
C-terminus (c)

Hass MA, Mulder FAA. Contemporary NMR Studies of Protein Electrostatics. Annu Rev Biophys. 2015;44:53-75.

* Additional pKa values
Acid 1 (J)
Acid 2 (U)
Acid 3 (Z)
Acid 4 (X)
Base 1 (B)
Base 2 (O)

For example, J=pSer, U=pThr, Z=pTyr. E.A. Bienkiewicz & K.J. Lumb, J Biomol NMR 15: 203-206 (1999).
Permanent negative charges can be added to the sequence by introducing acid J,U,Z, or X in the sequence and setting its pKa to -10; For permanent positive charges, include a base B or U in your sequence, with pKa=24.


Results

When the calculations are done, you will find the resulting pKa constants and Hill parameters for each titration site in the box below. You will also find graphical output: (1) deviation of pKa value for each site (2) total peptide charge as function of pH, (3) titration curves for each site. You can download each graph by clicking on it. In each graph the blue line represents the calculated charge at that site as a function of pH, the green curve is a fit to the Henderson-Hasselbalch equation, which includes a fitted pKa value and Hill parameter (nH: a measure of cooperativity) and in red the derivative of the blue curve, the "proton binding capacitance", which more clearly shows electrostatic coupling in the system. For further explanations, please see Lindman et al. Biochemistry 45, 13993-14002 (2006).