Structure determination of membrane proteins in five easy pieces

Rotational Alignment (RA) solid-state NMR provides the basis for a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. Membrane proteins are high priority targets for structure determination, and are challenging for existing experimental methods. Because membrane proteins reside in liquid crystalline phospholipid bilayer membranes it is important to study them in this type of environment. The RA solid-state NMR approach we have developed can be summarized in five steps, and incorporates methods of molecular biology, biochemistry, sample preparation, the implementation of NMR experiments, and structure calculations. It relies on solid-state NMR spectroscopy to obtain high-resolution spectra and residue-specific structural restraints for membrane proteins that undergo rotational diffusion around the membrane normal, but whose mobility is otherwise restricted by interactions with the membrane phospholipids. High resolution spectra of membrane proteins alone and in complex with other proteins and ligands set the stage for structure determination and functional studies of these proteins in their native, functional environment.     

Four easy pieces: mechanisms underlying circadian regulation of growth and development

The circadian clock confers rhythms of approximately 24 hours to biological events. It elevates plant fitness by allowing plants to anticipate predictable environmental changes and organize life process to coincide with the most favorable environmental conditions. Many developmental events are circadian regulated to ensure that growth occurs at the ideal time or season relative to available resources. Circadian clock control over growth and development is often achieved through regulation of key phytohormone action. Circadian influence over the genome is widespread and the clock modulates genes involved in phytohormone synthesis and signaling, in addition to other pathways shaping growth and development. This review presents four nonmutually exclusive mechanisms by which temporal information is gleaned from the core oscillator and passed to pathways regulating plant growth and development.     

Assembling the pieces

Transition metals function as cofactors in specific proteins, catalyzing electron exchange reactions, binding substrates and stabilizing protein structure. Studies of human diseases and of model organisms have defined many of the molecular details of metal uptake, trafficking, and excretion. The current challenge is to integrate these details into a systematic view of metal content, speciation, localization and use within organisms and ecosystems.     

HIF-1 regulation: not so easy come, easy go

The hypoxia-inducible factor-1 (HIF-1) is the master regulator of the cellular response to hypoxia and its expression levels are tightly controlled through synthesis and degradation. It is widely accepted that HIF-1alpha protein accumulation during hypoxia results from inhibition of its oxygen-dependent degradation by the von Hippel Lindau protein (pVHL) pathway. However, recent data describe new pVHL- or oxygen-independent mechanisms for HIF-1alpha degradation. Furthermore, the hypoxia-induced increase in HIF-1alpha levels is facilitated by the continued translation of HIF-1alpha during hypoxia despite the global inhibition of protein translation. Recent work has contributed to an increased understanding of the mechanisms that control the translation and degradation of HIF-1alpha under both normoxic and hypoxic conditions.     

Transfer RNA genes in pieces

The short genes encoding transfer RNA (tRNA) molecules are highly conserved in both sequence and structure, reflecting the central role of tRNA in protein biosynthesis. The frequent occurrence of fragmented intron-containing tRNAs that require processing to form contiguous molecules is therefore surprising. Recent discoveries of permuted and split tRNA genes have added to the apparent creativity of nature regarding the organization of these fragmented genes. Here, we provide an overview of the various types of fragmented tRNA genes and examine the hypothesis that the integration of mobile genetic elements--including viruses and plasmids--established such genes in pieces.     

Bacterial ribosomal RNA in pieces

The exact knowledge on the ribosomal RNA (rRNA) structure is an important prerequisite for work with rRNA sequences in bioinformatic analyses and in experimental research. Most available rRNA sequences of bacteria are based on gene sequences and on similarity analyses using Escherichia coli rRNA as a standard. Therefore, it is often overlooked that many bacteria harbour mature rRNA 'in pieces'. In some cases, the processing steps during the fragmentation lead to the removal of rRNA segments that are usually found in the ribosome. In this review, the current knowledge on the mechanisms of rRNA fragmentation and on the occurrence of fragmented rRNA in bacteria is summarized, and the physiological implications of this phenomenon are discussed.