The energetic profiles of a significant number of protein-DNA systems at 20°C reveal that, despite comparable Gibbs free energies, association with the major groove is primarily an enthalpy driven process, whereas binding to the minor groove is characterized by an unfavorable enthalpy that is compensated by favorable entropic contributions. These distinct energetic signatures for major versus minor groove binding are irrespective of the magnitude of DNA bending and/or the extent of binding-induced protein refolding. The primary determinants of their different energetic profiles appear to be the distinct hydration properties of the major and minor grooves, namely that the water in the AT-rich minor groove is in a highly ordered state and its removal results in a substantial positive contribution to the binding entropy. Since the entropic forces driving protein binding into the minor groove are a consequence of displacing water ordered by the regular arrangement of polar contacts, they cannot be regarded as hydrophobic. KeywordsDNA binding; DNA grooves; hydration; thermodynamics; electrostatics Structural and energetic characterizations of protein-nucleic acid complexes are important for a better understanding of the molecular interactions that govern transcriptional regulation. Of particular importance are the energetic profiles of DNA binding domains (DBDs) interacting with their target recognition sites. DBDs are known to interact specifically with either the major or minor grooves of DNA, with binding-induced structural effects ranging from negligible perturbation of the B-DNA conformation to substantial distortions, such as bending and kinking. One can then ask if there are qualitative differences in the forces driving protein binding to the different grooves of DNA. Comparing the association constants of these two types of DBDs does not furnish a satisfactory answer, since both categories contain examples of stronger and weaker binding interactions. An answer to this question therefore requires a detailed analysis of the forces involved in the formation of the specific protein-DNA complexes. This assumes not only measurement of the association constant but also determination of the Gibbs energy and its enthalpic and entropic components over a broad range of conditions, particularly temperature and ionic strength. In this review, we analyze the thermodynamic characteristics of protein binding to DNA published over the last several years. This overall consideration has revealed qualitative differences in the energetic signatures of

α-Synuclein (αS) is an amyloidogenic intrinsically disordered protein implicated in Parkinson's disease, for which copper-mediated pathways of neurodegeneration have been suggested. We have employed nuclear magnetic resonance, circular dichroism, electrospray ionization mass spectrometry, and thioflavin T fluorescence to characterize interactions of Cu(2+) with the physiological acetylated form (Ac-αS). Significantly, N-terminal acetylation abolishes Cu(2+) binding at the high-affinity M1-D2 site present in the nonacetylated protein and maintains Cu(2+) interactions around H50/D121. Fibrillation enhancement observed at an equimolar Cu(2+) stoichiometry with the nonacetylated model does not occur with Ac-αS. These findings open new avenues of investigation into Cu(2+)-mediated neurodegenerative pathology suggested in vivo.

DNA bulges are biologically consequential defects that can arise from template-primer misalignments during replication and pose challenges to the cellular DNA repair machinery. Calorimetric and spectroscopic characterizations of defect-containing duplexes reveal systematic patterns of sequence-context dependent bulge-induced destabilizations. These distinguishing energetic signatures are manifest in three coupled characteristics, namely: the magnitude of the bulge-induced duplex destabilization (ΔΔGBulge); the thermodynamic origins of ΔΔGBulge (i.e. enthalpic versus entropic); and, the cooperativity of the duplex melting transition (i.e. two-state versus non-two state). We find moderately destabilized duplexes undergo two-state dissociation and exhibit ΔΔGBulge values consistent with localized, nearest neighbor perturbations arising from unfavorable entropic contributions. Conversely, strongly destabilized duplexes melt in a non-two-state manner and exhibit ΔΔGBulge values consistent with perturbations exceeding nearest-neighbor expectations that are enthalpic in origin. Significantly, our data reveal an intriguing correlation in which the energetic impact of a single bulge base centered in one strand portends the impact of the corresponding complementary bulge base embedded in the opposite strand. We discuss potential correlations between these bulge-specific differential energetic profiles and their overall biological implications in terms of DNA recognition, repair and replication.

SUMMARY Targeting of type III secretion proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone CesAB to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB, but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB–EspA complex and EscN resulted in severe secretion and infection defects. We further show that the targeting and secretion signals are distinct and the two processes are likely regulated by different mechanisms.

Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N6-(2-Deoxy-α,β-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme involves the synthesis of an oligonucleotide precursor containing a 5-nitropyrimidine moiety at the desired lesion site via standard solid-phase procedures. Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation and subsequent formylation. NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals the presence of rotameric and anomeric species. The latter have been characterized by synthesizing the tridecamer oligodeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol to resolve the isomeric components. Hybridization of the chromatographically isolated fractions with their complementary d(CGCATGCGTACGC) counterpart yields two Fapy-dG·C duplexes that are differentially destabilized relative to the canonical G·C parent. The resultant duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of α- and β-anomers, the former more destabilizing than the latter. These anomer-specific impacts are discussed in terms of differential repair enzyme recognition, processing and translesion synthesis.