Quantifying Functional Group Interactions That Determine Urea Effects on Nucleic Acid Helix Formation

Abstract

Urea destabilizes helical and folded conformations of nucleic acids and proteins, as well as protein-nucleic acid complexes. To understand these effects, extend previous characterizations of interactions of urea with protein functional groups, and thereby develop urea as a probe of conformational changes in protein and nucleic acid processes, we obtain chemical potential derivatives (μ23 = dμ2/dm3) quantifying interactions of urea (component 3) with nucleic acid bases, base analogs, nucleosides and nucleotide monophosphates (component 2) using osmometry and hexanol-water distribution assays. Dissection of these μ23 yields interaction potentials quantifying interactions of urea with unit surface areas of nucleic acid functional groups (heterocyclic aromatic ring, ring methyl, carbonyl and phosphate O, amino N, sugar (C,O)); urea interacts favorably with all these groups, relative to interactions with water. Interactions of urea with heterocyclic aromatic rings and attached methyl groups (as on thymine) are particularly favorable, as previously observed for urea-homocyclic aromatic ring interactions. Urea m-values determined for double helix formation by DNA dodecamers near 25°C are in the range 0.72 to 0.85 kcal mol−1 m−1 and exhibit little systematic dependence on nucleobase composition (17–42% GC). Interpretation of these results using the urea interaction potentials indicates that extensive (60–90%) stacking of nucleobases in the separated strands in the transition region is required to explain the m-value. Results for RNA and DNA dodecamers obtained at higher temperatures, and literature data, are consistent with this conclusion. This demonstrates the utility of urea as a quantitative probe of changes in surface area (ΔASA) in nucleic acid processes.

Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes

“…For these predictions, carbonyl O, phenolic hydroxyl O and amino N of the aromatic compounds are treated as amide O, hydroxyl O and amide N, respectively. α -Values for interactions of urea with carbonyl O and amino N of nucleobases are similar though not identical to α-values for interactions of urea with amide O and amide N of protein model compounds 61 . For the 16 non-steroid compounds (derivatives of benzene, naphthalene and biphenyl) agreement between predicted and observed µ 23 values is generally within ±25%, and is not significantly different for the ten compounds with carbonyl O, phenolic hydroxyl O, and/or amino N than for the six compounds lacking these groups.…”

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

“…An example is the mechanism of open complex formation and stabilization in transcription initiation by Escherichia coli RNA polymerase (RNAP) at a DNA promoter (47). Here, information about interactions of solutes and noncoulombic (Hofmeister) interactions of salt ions with nucleic acid functional groups is also needed; this has been obtained for urea (11,12), and research with other solutes and salts is in progress. The small effects of urea and Hofmeister salts on the rate and equilibrium constants for the step involving opening of 13 bp of duplex promoter DNA provide evidence that RNAP opens this region in the active site cleft (14,48,49).…”

“…Recently, the interactions of key denaturants, osmolytes, and Hofmeister salts with the functional groups of proteins and nucleic acid have been determined, allowing these solutes to be used as thermodynamic and kinetic-mechanistic probes of the amount and the composition of the surface buried or exposed in the steps of a noncovalent protein or nucleic acid process like helix formation, folding, or binding (6)(7)(8)(9)(10)(11)(12)(13)(14). For protein folding, urea and guanidinium chloride (GuHCl) thermodynamic m-values (derivatives of the standard free energy of folding with respect to denaturant concentration) are determined almost entirely by the preferential interactions of these denaturants with the hydrocarbon and amide surface buried in folding (6):…”

Aspects of Weak Interactions between Folate and Glycine Betaine

“…70−72 This suggests that it will likely be difficult to predict μ 23 / RT values for proteins that release and/or take up protons upon binding, undergo dynamic motion, or use “wet interfaces” for binding, for intrinsically disordered sequences and for protein folding, although the Record lab has had some success with the latter case. 21,73 …”