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A double-deletion method to quantifying incremental binding energies in proteins from experiment: example of a destabilizing hydrogen bonding pair.

Abstract：

:The contribution of a specific hydrogen bond in apoflavodoxin to protein stability is investigated by combining theory, experiment and simulation. Although hydrogen bonds are major determinants of protein structure and function, their contribution to protein stability is still unclear and widely debated. The best method so far devised to estimate the contribution of side-chain interactions to protein stability is double mutant cycle analysis, but the interaction energies so derived are not identical to incremental binding energies (the energies quantifying net contributions of two interacting groups to protein stability). Here we introduce double-deletion analysis of 'isolated' residue pairs as a means to precisely quantify incremental binding. The method is exemplified by studying a surface-exposed hydrogen bond in a model protein (Asp96/Asn128 in apoflavodoxin). Combined substitution of these residues by alanines slightly destabilizes the protein due to a decrease in hydrophobic surface burial. Subtraction of this effect, however, clearly indicates that the hydrogen-bonded groups in fact destabilize the native conformation. In addition, molecular dynamics simulations and classic double mutant cycle analysis explain quantitatively that, due to frustration, the hydrogen bond must form in the native structure because when the two groups get approximated upon folding their binding becomes favorable. We would like to remark that 1), this is the first time the contribution of a specific hydrogen bond to protein stability has been measured by experiment; and 2), more hydrogen bonds need to be analyzed to draw general conclusions on protein hydrogen bond energetics. To that end, the double-deletion method should be of help.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Campos LA,Cuesta-López S,López-Llano J,Falo F,Sancho J

doi

10.1529/biophysj.104.050203

subject

Has Abstract

pub_date

2005-02-01 00:00:00

pages

1311-21

issue

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(05)73197-0

journal_volume

pub_type

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A double-deletion method to quantifying incremental binding energies in proteins from experiment: example of a destabilizing hydrogen bonding pair.