BIOCHEMISTRY - DR. JAKUBOWSKI
Last Update: 3/2/16
Learning Goals/Objectives for Chapter 2F: After class and this reading, students will be able to ...
In the last decade, the contributions to the overall stability of a protein from the hydrophobic effect and H bonds has been studied using site specific mutagenesis. In this technique, the DNA coding sequence for a given amino acid in a gene can be altered so that the new mutant protein differs from the normal protein (often called the wild type protein) by one amino acid. To probe the hydrophobic effect, for example, a buried hydrophobic amino acid like Ile could be changed to Gly which is much smaller, and offers less hydrophobic contribution to the stability of the native state. The result of this mutation might leave a "hole" in the protein (not unlike the vacant holes in crystal structures of salts). This "hole" might be diminished in size by subtle rearrangement of the protein structure in the vicinity. Certain amino acids would not be used as replacements in such studies. For instance, an Ile would not be replaced with a positively charge Arg which would clearly destabilize the protein. The extent of destabilization in mutant proteins can be determined by calculating the DGo for the native to denatured transition using urea as the denaturing agent.
Previously, the following statistics were presented concerning the distribution of amino acids in the tertiary structure of a protein. New values are shown below in red, based on much more crystallographic data, as summarized in Pace's article.
Two articles by Pace suggests that Dills "influential review (from which much of the above derives) that concluded that hydrophobicity is the dominant force in protein folding" should be rethought. Using site specific mutagenesis to change Asn (which can H bond through its side chain) to Ala (which can't) in a variety of proteins, he has shown that approximately 80 cal/mol/A3 of stability is gained if a side chain (in this case Asn) can form buried H bonds to buried amide links of the protein backbone. Similar studies of mutants in which Leu is replaced with Ala, and Ile with Val, suggests that only 50 cal/mol/A3 is gained from burying a hydrophobic -CH2- methylene group. Extending these results to protein folding suggest that proteins stability is determined more by the formation of buried H bonds than by the hydrophobic effect.
The investigators measured DGo for the N <=> D transition (presumably by varying the urea concentration and extrapolating the DGo for unfolding to 0 M urea (see: Lab Determination of DGo of Unfolding). For the reaction as written, DGounfolding > 0 at room temperature and 0 M urea. The mutant protein, since they are destabilized, would have a less positive value for DGounfolding (They would also have a less negative value for folding since they are less stable). The difference in DGounfolding between the wild type and mutant (DDG) is expressed as:
DDG = DGounfolding wild-type - DGounfolding mutant > 0
DDG > 0 since DGounfolding wild-type > DGounfolding mutant. The more positive the DDG, the more the mutant is destabilized in comparison to the wild type. The data for a series of mutants is shown below.
Analysis of Mutants: H Bonds in Protein Folding
|mutation||DVol side chain (A3)||% buried||DDG (kcal/mol)
|Asn to Ala||37.4||95||2.9||78|
|Leu to Ala||74.5||99||3.6||48|
|Ile to Val||25.8||100||1.3||50|
What leads to protein stabilization/destabilization when Asn is changed to Ala?
One possible contributor to stability is the side chain conformational entropy. Since in the mutant the Ala would find itself in a larger "hole" and have greater freedom for motion, it would have more conformational entropy that would stabilize the mutant over the wild type. Hence this effect can NOT explain the observed destabilization of the Asn to Ala mutant.
In the proteins he studied, only one of eight Asn to Ala mutation involved an Asn in a helix, so the average change could not be attributable to differences in helix propensities for the two amino acids.
In the mutants, assuming no rearrangement of the remaining side chains, there is an "unnecessary" and unoccupied 37.4 A3 cavity. To create this cavity is thermodynamically unfavorable (about 22 cal/mol/A3 obtained from values for hydrophobic mutations). If the same penalty were applied here, the Asn to Ala mutant would be destabilized by 0.8 kcal/mol (22 x 37.4), This is significantly less that the observed destabilization (2.9 kcal/mol), so this effect also could not account for the destabilization of the Asn to Ala mutants.
If there were compensatory changes to minimize the cavity size, this would only help to stabilize the protein and hence can not account for the observed destabilization.
Possible Explanation of Destabilization of Asn to Ala Mutants
|possible reasons||explanation||effect on mutant||support observed destab. of mutant?|
|residue conformational entropy||Ala in bigger hole:
more freedom motion;
free energy change
energy penalty to make an unoccupied cavity
approx. 0. 8 kcal/mol
yes but of insufficient size compared to the observed effect (2.9 kcal/mol
|free energy change
protein conformational changes
|rearrange protein to fill cavity||stabilize mutant||NO|
Hence these alternative sources to explain the destabilization of the mutant can't account for the data and we're left with the explanation that the stability of the native protein over the mutant is accounted for by burying the H bond donor and acceptors of the amide group and associated changes in van der Waals interactions.
Pace argues that burying the amide group of Asn is similar to burying the peptide bond of the main chain. There sizes are very comparable. Free amide groups can form four H bonds, but peptide (amide) groups can only form three. Even if the value of 78 for the DDG (cal/mol/A) is adjusted for this, the new value of 62 is still larger than that for burying a methylene group. Analysis of 108 folded proteins has shown that hydrophobic groups contribute 118,200 A3 of buried volume, compared to 92,000 A3 for peptide groups. Multiplying these figures by 78 and 49 (from the above table) suggests that overall, burying peptide groups contributes more to protein stability than burying hydrophobic groups.
Would electrostatic interactions of the buried peptide group with the surrounding environment destabilize a protein? Pace argues that this would be more than compensated for by favorable van der Waal's interactions (short range) at the buried site. This can be illustrate by comparing the DG transfer of an amide from water to the vapor (11.2 kcal/mol)) compared from water to cyclohexane (7.6 kcal/mol). Transfer to the vapor is more unfavored (due to the desolvation required when it moves to the gas phase) than to cyclohexane, even though a cavity must be created in the cyclohexane (a process which would be unfavored entropically). Transfer to octanol is even more favored (1.4 kcal/mol) but all these values are still positive (disfavored). Similar experiments with transfer of a methylene group (-CH2-) are negative, given the hydrophobic effect and the close packing van der Waal's interactions possible. These suggest that van der Waals interactions formed on burying an amide in any solvent are stabilizing. Now consider the packing density of atoms for various substances:
|closest packed spheres||0.71|
From this table it should be apparent that van der Waals interactions (short range) will be more stabilizing in the interior of the protein compared to the same groups in bulk water (or in the denatured state). Carbonyl groups are more polarizable than methylene groups, which should contribute to van der Waals interactions.
One other addition. It has been noted that Gly peptides are not very soluble in water. The backbone, even with the polar peptide bonds appears to be solvophobic. If the backbone of any polymer can't interact well with the solvent - i.e. the solvent is "poor" - then the backbone interacts with itself, which drives collapse. If the backbone interacts well with a "good" solvent, it won't collapse as readily.
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