Biochemistry Online: An Approach Based on Chemical Logic

Biochemistry Online





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

  • describe the differences between primary, secondary, supersecondary, tertiary, quaternary and domain protein structure
  • explain the basis of CD measurements for secondary structure
  • describe the similarities between torsion angles and an energy vs torsion angle plot for the rotation of the C2-C3 torison angle with phi/psi angles of peptide bonds and the 2D plots off allowed conformations around a given amino acid in a protein (Ramachandran plot).
  • (from reading give explanation for observed propensities of amino acids for different secondary structure)

C3.  Tertiary Structure

To a first approximation, you may think the a globular protein might fold such thal all the hydrophobic side chains are buried in the interior of the protein, surrounded by other hydrophobic side chains.  In a similar fashion, you might expect the polar and charged side chains could be on the surface, exposed to water.  Such a model would be analogous to a micelle which has an almost "perfect" separation of polar (on the surface) and nonpolar atoms (buried).  If it were only that simple!.  Topologically, it is impossible for a protein to fold in an intramolecular fashion in strict analogy to the the intermolecular aggregation of single chain amphiphiles into a micelle.   Consider also that the entire backbone is polar.  To a first approximation we would expect the bulk nonpolar groups would be buried surrounded by other nonpolar groups.  Likewise we would expect the bulk of polar and charged would be on the surface.  The Jmol models below show the similarities in the formations of a micelle, in which all nonpolars are buried, to that of protein in which most nonpolar side chain are buried and surrounded in a nonpolar environment.

Jmol:  Updated  A protein with a buried nonpolar amino acid Jmol14 (Java) |  JSMol  (HTML5)       

JmolUpdated Micelle  Jmol14 (Java) |  JSMol  (HTML5) 


What is the preferred disposition of side chains in proteins as evidenced from the crystal structure of thousands of proteins?  Here are some conclusions from a paper by  Pace (Biochemistry. 40, pg 310 (2001)

Tertiary structure and pKas

 If a charged size chain is buried in a protein, you would expect that it would be surrounded, in general, by either oppositely charged side chains, to which it could form an internal salt bridge (ion-ion interaction), or a polar uncharged group with which it could interact through dipole-dipole or, more specifically, H bond interactions.  You would also expect that if it were not near an oppositely charged side chain, that it would exist, if buried, in an uncharged state. 

Hence the pKa of side chains would be dramatically affected by the nature of its microenvironment (as we have already seen with the pKa of acetic acid in solvents of different polarity).  NMR spectroscopy has been used to determine the pKa values of specific side chains in protein whose crystal structure is known.  Pace et al (2009) summarize data on the properties of ionizable side chains in a series of proteins whose structure has been determined. The intrinsic pKa, pKaint or prototypical pKa value for a side chain exposed to water can be determined using  a pentapeptide containing the target amino acid X surrounded by 2 Ala ion either each side with both the N and C termini of the peptide blocked so they are uncharged.

Table:  pKa values of ionizable side chains in a series of protein

Group Content % Buried % pKa int in AAXAA pKa avg low pKa high pKa  # measurments
Asp 5.2 56 3.9 3.5 + 1.2 0.5 9.2 139
Glu 6.5 48 4.3 4.2 + 0.9 2.1 8.8 153
His 2.2 72 6.5 6.6 + 1.0 2.4 9.2 131
Cys 1.2 90 8.6 6.8 + 2.7 2.5 11.1 25
Tyr 3.2 67 9.8 10.3 + 1.2 6.1 12.1 20
Lys 5.9 34 10.4 10.5 + 1.1 5.7 12.1 35
Arg 5.1 56 12.3        
C term     3.7 3.3 + 0.8 2.4 5.9 22
N term     8.0 7.7 + 0.5 6.8 9.1 16

A quick glance a the table shows a huge variation in the pKas of ionizable side chains in proteins with the pKa of Asp varying over a range of 8.7 pH units, showing that it can act at physiological pH as either a strong acid or a moderate base.   Three majors effects can perturb the pKa of ionizable side chains:

1.  Dehydration of side chain as it is buried in a protein (Born Effect): The stability of a charged group depends on the polarity of the medium in which it exists.  Ions are more stable in water than in nonpolar solvents as the water molecules can reorient and interact with the ion through ion-dipole or ion-H bond interactions, which effectively shields the ion from other counter ion.  The shielding effect of water is related to the dielectric constant, e, of the solvent.   Coulombs law can be written as Fcoul = q1q2/er2 which can be expressed in energy terms as DGcoul = q1q2/er.   Epsilon is the dielectric constant of the solvent.  Water has a higher dlelectric constant (80) than nonpolar solvents (4-10) and hence shields opposing charges more, stabilizing them.  Hence the pKa of side chains of those amino whose deprotonated state is charged will  have their pKa values raised (so they are less acidic) in nonpolar environments. The reverse holds for side chains whose protonated form is charged.  Pace cites as an example two mutant of staphylococcal nuclease in which a buried Val 66 is changed either to Asp or Lys.  The buried Asp has a pKa of 8.9 compared to  5.5 for the buried Lys.  These changes were not compensated for with new charge-charge interactions, so the change can be attributed to the dehydration (or Born) effect.

2.  Ion-Ion interactions with another charged side chain through Coulombic forces:  This effect can be most readily observed at the surface of the protein.  Pace cites a study of RNase Sa that is devoid of Lys and has a pI of 3.5.  Five Asp and Glu were replaced on the surface using site-specific mutagenesis with Lys, which changed the pI of the protein to 10.2.    At pH 7, the protein without Lys had a charge of -7 while the protein with 5 Lys had a charge of +3.  The crystal structures were similar so Coulombic interactions would determine the differences in the pKa of the 11 common side chains.   On average the mutant pKas were higher by 0.75  pH units, which makes sense as the mutant had a high pI.   Calculated pKa values were  similar to those determined by NMR.  These data are consistent with the idea that Coulombic interactions are the chief cause of pKa changes in surface side chains.

3.  Charge-dipole interactions and H bonds:  It should be obvious that charge states of ionizable side chains would be adjusted to optimize H bond (and more generally charge-dipole) interactions in proteins.  If the interactions are optimal in the charged state, pKa values for His and Lys would be increased and for Asp, Glu, Cys, and Tyr they would be decreased.   Pace cites the buried Asp 76 in RNase T1 in which the Asp is charged but does not form an internal salt bridge.  It has a depressed pKa of 0.6 and has 3 H bonds to the side chains of Asn 9, Tyr 11 and Thr 91.    Mutants were made to remove the H bonds to see the effect on the pKa of Asp 76.   Removing 1, 2, or 3 H bonds changed the pKa to 3.3, 5.1, and 6.4 respectively.  The 6.4  value is much higher than the pKint, which can be attributed to the Born effect.


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