Descripción: Since the elucidation of the myoglobin (Mb) structure, a histidine residue on the E helix (His-E7) has been proposed to act as a gate with an open or closed conformation controlling access to the active site. Although it is believed that at low pH, the His-E7 gate is in its open conformation, the full relationship between the His-E7 protonation state, its conformation, and ligand migration in Mb is hotly debated. We used molecular dynamics simulations to first address the effect of His-E7 protonation on its conformation. We observed the expected shift from the closed to the open conformation upon protonation, but more importantly, noted a significant difference between the conformations of the two neutral histidine tautomers. We further computed free energy profiles for oxygen migration in each of the possible His-E7 states as well as in two instructive Mb mutants: Ala-E7 and Trp-E7. Our results show that even in the closed conformation, the His-E7 gate does not create a large barrier to oxygen migration and permits oxygen entry with only a small rotation of the imidazole side chain and movement of the E helix. We identify, instead, a hydrophobic site in the E7 channel that can accommodate an apolar diatomic ligand and enhances ligand uptake particularly in the open His-E7 conformation. This rate enhancement is diminished in the closed conformation. Taken together, our results provide a new conceptual framework for the histidine gate hypothesis. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: Genomes contain the necessary information to ensure that genes are expressed in the right place, at the right time, and with the proper rate. Metazoan developmental genes often possess long stretches of DNA flanking their coding sequences and/or large introns which contain elements that influence gene expression. Most of these regulatory elements are relatively small and can be studied in isolation. For example, transcriptional enhancers, the elements that generate the expression pattern of a gene, have been traditionally studied with reporter constructs in transgenic animals. These studies have provided and will provide invaluable insights into enhancer evolution and function. However, this experimental approach has its limits; often, enhancer elements do not faithfully recapitulate native expression patterns. This fact suggests that additional information in cis-regulatory regions modulates the activity of enhancers and other regulatory elements. Indeed, recent studies have revealed novel functional aspects at the level of whole cis-regulatory regions. First, the discovery of "shadow enhancers." Second, the ubiquitous interactions between cis-regulatory elements. Third, the notion that some cis-regulatory regions may not function in a modular manner. Last, the effect of chromatin conformation on cis-regulatory activity. In this article, I describe these recent findings and discuss open questions in the field. © 2012 Wiley Periodicals, Inc.