Biochemistry Online: An Approach Based on Chemical Logic

Biochemistry Online




Last Update:  3/2/16

Learning Goals/Objectives for Chapter 2F:  After class and this reading, students will be able to ...

  • Differentiate between general charge and specific ion-ion pairs and summarize their role in protein stability
  • Draw the structure of N-methylacetamide  (NMA) and explain why it is a useful small molecule model to study the role of H bonds in protein stability
  • Draw a thermodynamics cycle for the transfer of a hydrogen bonded dimer of NMA from water to a nonpolar environment.  From the DG0 for steps in the cycle, and extending this model to protein, predict if buried H bond formation drives protein folding
  • Explain if studies of low temperature protein denaturation, high temperature protein, and DGo transfer of nonpolar side chains from water to more nonpolar solvents support the hydrophobic effect in protein stability
  • summarize the relationship between the empirical Hofmeister series and preferential binding of reagents into the hydration sphere of protein to explain the effects of denaturants (urea, guanidine salts) and stabilizers (glycerol, ammonium sulfate) on proteins
  • Using benzene solubility in water as a model to study the role of hydrophobic effect in protein unfolding and by inference in protein stability, interpret graphs of DG0, DH0, DS0 and DCp for the transfer of benzene to water, as a function of temperature.

  • from the above graph, explain if trends in the thermodynamic parameters for benzene transfer into water predict the observed protein unfolding/stability behavior of proteins as a function of temperature?

  • Give a molecular interpretation of the observed DCp for the transfer of nonpolar molecules into water.

  • Describe chain conformational entropy, relate it to conformational changes in acyl side chains in single and double chain amphiphiles with temperatures, and describe it role in protein stability.

  • state which of several given explanations for the observed destabilizing effects of Asn to Ala mutations in protein account for those observation

  • summarize graphically the magnitude and direction of the major contributors (inter- and intramolecular forces and effects) to protein stability

F9.  Protein Stability and Molecular Orbitals

A more complete description of the energetics underlying protein structure and stability would involve quantum mechanical effects.  These have not been studied in detail since quantum mechnanical calculations are difficult to perform on large molecules.  Recent work has led an understanding of a new source of stability of proteins based on overlap of proximal molecular orbitals that stabilizes a protein through electron delocalization.  Two common examples of electron delocalization that increase stability are  resonance and hyperconjugation.   Resonance involves delocalization of electrons through overlap of adjacent atomic p orbitals to form low energy molecular bonding orbitals.  You may remember hyperconjugation being used to explain the relative stability of tertiary carbocations compared to secondary or primary ones.  The typical explanation involves electron donation and pushing from the adjacent methyl groups which effectively decreases the charge on the carbocation.  A more correct explanation involves overlap of molecular orbitals, specifically of a C-H  s orbital from one of the C-H bonds of the methyl group with the partially filled p orbital on the carbon atom containing the charge as seen in the figure below. 

Figure:  Stabilization of Carbocations by Hyperconjugation

It should also not be surprising that orbital overlap accounts for hydrogen bonds interactions (in contrast to the more simplistic explanation of the attractions between the partial charges on hydrogen bond donors and acceptors).  Bartlett et al (2010) have recently described two types of orbital overlaps involving electron delocalization that stabilize protein structures.  Molecular orbital description show that two lone pairs on the oxygen (hydrogen bond donor) in amide bonds are different.  One pair, ns (Figure a below) is in a nonbonding orbital with 60% s character, while the other, np (figure b below), has approximately 100% p character.    Figure c below shows that a hydrogen bond between the carbonyl oxygen of the ith amino acid and the amide H of the ith+4 amino acid can be described in quantum mechanical terms as the overlap (i.e. delocalization) of the ns nonbonding orbital on oxygen and the  s* antibonding N-H orbital.  What about the np electrons on the carbonyl O?  They found evidence for a new kind of stabilizing influence in proteins as the np orbital overlaps with the an antibonding p* orbital on the ith+1 carbonyl group (figure d below) when the two amino acids are proximal as parts of secondary structures, including alpha and 310 helices as well as in twisted b sheets.

Figure:  Orbital involvement in H-bonds and n to pi* interactions in alpha helices

(a) s-rich lone pair of an amide oxygen. (b) p-rich lone pair of an amide oxygen. (c) ns to σ*: hydrogen bond in an a-helix. (d) np to p*: n to p* interaction in an a-helix.  Reprinted by permission from Macmillan Publishers Ltd: Nature Chemical Biology.  Bartlett, G. et al. n to pi* interactions in protein.  6, pg 615 (2010)

To test which phi-psi angles would allow close enough approach for np to p* interactions, the investigators performed quantum mechanical computations on possible conformations of AcAla4NHMe for which np to p* were possible (see figure A below).  These calculations showed these interaction were possible for values of d ≤ 3.2 � and 99� ≤ θ ≤ 119� (figure a below).  Distances less than 3.2 angstroms, van der Waals surfaces of the C and O on adjacent carbonyls overlap, suggesting that the interaction is quantum mechanical and not classically mediated.   Next they calculated d and q values for on high resolution x-ray structures of known proteins.    Red shading in the Ramachandran plot in figure c below phi/psi regions in actual proteins that meet the required geometry for  np to p*  overlap.  Hence these geometry are abundant in proteins, especially in secondary structures described above. 

Figure:  Ramachandran plots of n to pi* interactions.


(a) Definitions of dihedral angles φ (C′i�1�Ni�Cα i�C′i) and ψ (Ni�Cαi�C′i�Ni+1), distance (d) and planar angle θ. Criteria for an n to p* interaction in the crystallographic analyses: d ≤ 3.2 �; 99� ≤ θ ≤ 119�. (b) Computational data showing the energy.  Reprinted by permission from Macmillan Publishers Ltd: Nature Chemical Biology.  Bartlett, G. et al. n to pi* interactions in protein.  6, pg 615 (2010)


Return to Chapter 2F: Thermodynamics and IMFs of Protein Stability

Return to Biochemistry Online Table of Contents

Archived version of full Chapter 2F: Thermodynamics and IMFs of Protein Stability


Creative Commons License
Biochemistry Online by Henry Jakubowski is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.