Energy landscapes have been used to conceptually describe and model protein folding but have been difficult to measure experimentally, in large part because of the myriad of partly folded protein conformations that cannot be isolated and thermodynamically characterized. Here we experimentally determine a detailed energy landscape for protein folding. We generated a series of overlapping constructs containing subsets of the seven ankyrin repeats of the Drosophila Notch receptor, a protein domain whose linear arrangement of modular structural units can be fragmented without disrupting structure. To a good approximation, stabilities of each construct can be described as a sum of energy terms associated with each repeat. The magnitude of each energy term indicates that each repeat is intrinsically unstable but is strongly stabilized by interactions with its nearest neighbors. These linear energy terms define an equilibrium free energy landscape, which shows an early free energy barrier and suggests preferred low-energy routes for folding.

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that ⌬G°for unfolding depends linearly on TMAO concentration, and that the sensitivity of ⌬G°to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of ⌬G°on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.Keywords: Protein stability; protein folding; Notch ankyrin domain; Barnase; osmolytes Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that is found in several marine organisms containing elevated intracellular urea concentrations (Robertson 1966(Robertson , 1975Griffith et al. 1974). Numerous studies have investigated the effect of TMAO on proteins and described its stabilizing effects (Yancey and Somero 1979;Lin and Timasheff 1994;Jaravine et al. 2000). Yancey et al. (1982) showed, by gel filtration chromatography, that TMAO promotes folding of proteins into more compact forms. They further showed, by recovery of enzymatic activity, that TMAO promotes folding to specific, biologically relevant native states (Yancey et al. 1982). Wang and Bolen (1997) provided an explanation for the ability of TMAO to promote specific refolding to the native structure, in which unfavorable thermodynamic interactions between TMAO and the peptide backbone destabilize the denatured state, shifting equilibrium toward the native state. Preferential interaction data from Lin and Timasheff (1994) are consistent with this interpretation, showing the region of solvent near the denatured state of the protein to be rarified in TMAO.The functional dependence of protein stability on TMAO has been analyzed in several systems and has led to different Reprint requ...

Might DNA sequence variation reflect germline genetic activity and underlying chromatin structure? Using two strains of medaka (Japanese killifish, Oryzias latipes), we compared genomic sequence and mapped ~37.3 million nucleosome cores from medaka Hd-rR blastulae, together with 11,654 representative transcription start sites from six embryonic stages. We observed a ~200-bp periodic pattern of genetic variation downstream of transcription start sites; the rate of insertions and deletions longer than 1bp peaked at positions approximately +200, +400, and +600bp, while the point mutation rate showed corresponding valleys. This ~200-bp periodicity was correlated with the chromatin structure, with nucleosome occupancy minimized at positions 0, +200, +400, and +600bp. These data exemplify the potential for genetic activity (transcription) and chromatin structure to contribute in molding the DNA sequence on an evolutionary timescale.Mutation and repair characteristics of DNA sequence in experimental systems have been shown in a number of cases to reflect structures in chromatin. For one well-studied experimental system, UV-treated yeast (S. cerevisiae), repair rates for a set of DNA nucleosome core regions are lower than in the surrounding linker regions (1-4). Correlations between chromatin structure and mutation rates have also been suggested in analysis of human and yeast genomes *joint corresponding authors. One-sentence summary: Sequence variation in the DNA Japanese killifish, Oryzias latipes, shows a periodic pattern downstream of transcription start sites that is strongly correlated with chromatin structure.

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