Introduction

Welcome to the protein m-value calculator.  This site permits the prediction of m-values for osmolyte-induced protein folding and unfolding.  The m-value is an experimentally determined quantity derived from the Linear Extrapolation Method, and is a measure of the osmolyte efficacy in folding or unfolding a protein.  It is also a measure of the cooperativity of folding/unfolding in the presence of the osmolyte, and it arises from the free energy contributions of protein groups that either become exposed upon unfolding or buried upon folding the protein.  The m-value has units of kcal/mol/M and it is positive for protecting osmolytes, such as TMAO or sarcosine, that drive the equilibrium toward the folded state.  If the osmolyte is urea, which drives the equilibrium toward the unfolded state, the m-value is negative.

This site uses the transfer free energy model to predict the m-value.  The m-value calculator first determines from the solvent accessible surface area the number of all side chains (sc) and backbone (bb) units exposed in the native structure. Multiplying the number of exposed sc and bb units by their experimentally determined transfer free energies and summing gives the transfer free energy of the native state from water to osmolyte solution. The transfer free energy of the denatured state is determined in like manner for three models of the denatured state: (1) a random coil in a good solvent (upper bound model), (2) a compact denatured state (lower bound model), and (3) a self avoiding random coil (Schellman model). M-values are obtained from the difference between the unfolded and native state transfer free energies for the three denatured state models and presented in the output.  A detailed method describing the calculation of m-values has been published in Auton, M & Bolen, DW, Methods Enzymol 428, 397-418 (2007).

Instructions

The m-value calculation is strictly applicable only to monomeric proteins that do not contain a cofactor or metal ion. Additionally, the calculated m-value applies to reversible two-state folding/unfolding conducted under conditions of neutral pH and moderate salt.  All that is required of the user of the site is to upload a PDB atomic coordinate file for the protein of interest.  Please note the following concerning input/output.

Input Caveats

  1. If the coordinates are given for multiple structures, the user must eliminate coordinates of all but one structure.  (NMR structure files contain many structures that represent part of the average solution structure.  A method to calculate the average m-value from multiple structures is currently in development.)
  2. If the monomeric protein crystallizes as a dimer, submit only the coordinates for the monomer.
  3. Sometimes the coordinates are not recognized properly as the standard PDB format may change.  Email Matthew Auton (mtauton@gmail.com) for questions about troubleshooting.)

Output

  1. The calculated m-value that corresponds best to the experimental m-value is the one using the self-avoiding random coil model (i.e. Schellman's model). M-values of upper and lower bound unfolded state models are also given.
  2. Transfer free energies of the native state from water to1M osmolyte solution, dissected into side chain and backbone energetic contributions are given.
  3. Transfer free energies from water to 1M osmolyte solution of the three unfolded state models, separated into side chain and backbone energetic contributions are provided.
  4. Solvent accessible surface area changes for amino acid side chains and backbone on folding/unfolding are given.
  5. Transfer free energy contributions are given for side chains and backbone units exposed or occluded on folding/unfolding, divided by the corresponding change in accessible surface area for that type of group.  These data permit construction of the group transfer free energy display shown in:   
    • Auton, M & Bolen, DW, Proc Natl Acad Sci USA 102(42), 15065-15068 (2005) [PUBMED] [PDF]
    • Auton, M, Holthauzen, LM & Bolen, DW, Proc Natl Acad Sci USA 104(39), 15317-15322 (2007). [PUBMED] [PDF]



 

Citing

Please Reference

If you would like to include our calculated m-value in your publications, please reference our methods paper:
Auton, M & Bolen, DW, Methods Enzymol 428, 397-418 (2007). PUBMED; PDF.