A Glimpse into the Structural Properties of the Intermediate and Transition State in the Folding of Bromodomain 2 Domain 2 by Φ Value Analysis

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site-directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C-terminal region represents part of the initial folding nucleus, while the N-terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native-like interactions have been identified, suggesting the presence of a non-compact, partially folded state with scarce native-like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non-native interactions that play a significant role in folding.

Single-molecule analysis of i-motif within self-assembled DNA duplexes and nanocircles

The cytosine (C)-rich sequences that can fold into tetraplex structures known as i-motif are prevalent in genomic DNA. Recent studies of i-motif–forming sequences have shown increasing evidence of their roles in gene regulation. However, most of these studies have been performed in short single-stranded oligonucleotides, far from the intracellular environment. In cells, i-motif–forming sequences are flanked by DNA duplexes and packed in the genome. Therefore, exploring the conformational dynamics and kinetics of i-motif under such topologically constrained environments is highly relevant in predicting their biological roles. Using single-molecule fluorescence analysis of self-assembled DNA duplexes and nanocircles, we show that the topological environments play a key role on i-motif stability and dynamics. While the human telomere sequence (C3TAA)3C3 assumes i-motif structure at pH 5.5 regardless of topological constraint, it undergoes conformational dynamics among unfolded, partially folded and fully folded states at pH 6.5. The lifetimes of i-motif and the partially folded state at pH 6.5 were determined to be 6 ± 2 and 31 ± 11 s, respectively. Consistent with the partially folded state observed in fluorescence analysis, interrogation of current versus time traces obtained from nanopore analysis at pH 6.5 shows long-lived shallow blockades with a mean lifetime of 25 ± 6 s. Such lifetimes are sufficient for the i-motif and partially folded states to interact with proteins to modulate cellular processes.

Molten Globule-Like Partially Folded State ofBacillus licheniformis α-Amylase at Low pH Induced by 1,1,1,3,3,3-Hexafluoroisopropanol

Effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) on acid-denaturedBacillus licheniformis α-amylase (BLA) at pH 2.0 was investigated by far-UV CD, intrinsic fluorescence, and ANS fluorescence measurements. Addition of increasing HFIP concentrations led to an increase in the mean residue ellipticity at 222 nm (MRE222 nm) up to 1.5 M HFIP concentration beyond which it sloped off. A small increase in the intrinsic fluorescence and a marked increase in the ANS fluorescence were also observed up to 0.4 M HFIP concentration, both of which decreased thereafter. Far- and near-UV CD spectra of the HFIP-induced state observed at 0.4 M HFIP showed significant retention of the secondary structures closer to native BLA but a disordered tertiary structure. Increase in the ANS fluorescence intensity was also observed with the HFIP-induced state, suggesting exposure of the hydrophobic clusters to the solvent. Furthermore, thermal denaturation of HFIP-induced state showed a non-cooperative transition. Taken together, all these results suggested that HFIP-induced state of BLA represented a molten globule-like state at pH 2.0.

AHSP (α-haemoglobin-stabilizing protein) stabilizes apo-α-haemoglobin in a partially folded state

To produce functional Hb (haemoglobin), nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. AHSP (α-haemoglobin-stabilizing protein) has been shown previously to bind αh and regulate redox activity of the haem iron. In the present study, we used a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh.