The experimental study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. l-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodology for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.

The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.

No abstract

Much recent literature explores controlled burning practices used by people of different cultures to manipulate landscapes. Because humans have only recently been able to suppress fires occurring at larger scales these studies focus on activities occurring at the scale of sites as making the greatest contribution to creating cultural landscapes. In this study we examine the role of fire in the construction of Anishinaabe cultural landscapes in the boreal forest of northwestern Ontario. Through our work with elders of Pikangikum First Nation we examined Anishinaabe knowledge and relationships to fire occurring across spatial and temporal scales. Pikangikum residents perceive forest fires as beings which possesses agency and who intentionally create order in landscapes. This notion suggests that cultural landscapes are more than the physical remains of the sum of human activities. The possibility of non-human agents having a role in the creation of meaningful spaces prompts us to call for a reassessment both of the scale of inquiry and the nature of cultural landscapes. We conclude with a discussion of the benefits and potential constraints to inclusion of indigenous cultural landscapes in current co-management arrangements.

Abstract. The Rara´muri (Tarahumara) people live in the mountains and canyons of the Sierra Madre Occidental of Chihuahua, Mexico. They base their subsistence on multiple-use strategies of their natural resources, including agriculture, pastoralism, and harvesting of native plants and wildlife. Pino Gordo is a Rara´muri settlement in a remote location where the forest has not been commercially logged. We reconstructed the forest fire regime from firescarred trees, measured the structure of the never-logged forest, and interviewed community members about fire use. Fire occurrence was consistent throughout the 19th and 20th centuries up to our fire scar collection in 2004. This is the least interrupted surface-fire regime reported to date in North America. Studies from other relict sites such as nature reserves in Mexico or the USA have all shown some recent alterations associated with industrialized society. At Pino Gordo, fires recurred frequently at the three study sites, with a composite mean fire interval of 1.9 years (all fires) to 7.6 years (fires scarring 25% or more of samples). Per-sample fire intervals averaged 10-14 years at the three sites. Approximately two-thirds of fires burned in the season of cambial dormancy, probably during the pre-monsoonal drought. Forests were dominated by pines and contained many large living trees and snags, in contrast to two nearby similar forests that have been logged. Community residents reported using fire for many purposes, consistent with previous literature on fire use by indigenous people. Pino Gordo is a valuable example of a continuing frequent-fire regime in a never-harvested forest. The Rara´muri people have actively conserved this forest through their traditional livelihood and management techniques, as opposed to logging the forest, and have also facilitated the fire regime by burning. The data contribute to a better understanding of the interactions of humans who live in pine forests and the fire regimes of these ecosystems, a topic that has been controversial and difficult to assess from historical or paleoecological evidence.