Troubleshooting Guide to Expressing Intrinsically Disordered Proteins for Use in NMR Experiments

Intrinsically disordered proteins (IDPs) represent a structural class of proteins that do not have a well-defined, 3D fold in solution, and often have little secondary structure. To characterize their function and molecular mechanism, it is helpful to examine their structure using nuclear magnetic resonance (NMR), which can report on properties, such as residual structure (at both the secondary and tertiary levels), ligand binding affinity, and the effect of ligand binding on IDP structure, all on a per residue basis. This brief review reports on the common problems and decisions that are involved when preparing a disordered protein for NMR studies. The paper covers gene design, expression host choice, protein purification, and the initial NMR experiments that are performed. While many of these steps are essentially identical to those for ordered proteins, a few key differences are highlighted, including the extreme sensitivity of IDPs to proteolytic cleavage, the ability to use denaturing conditions without having to refold the protein, the optimal chromatographic system choice, and the challenges of quantifying an IDP. After successful purification, characterization by NMR can be done using the standard 15N-heteronuclear single quantum coherence (15N-HSQC) experiment, or the newer CON series of experiments that are superior for disordered proteins

The Disordered Dehydrin and Its Role in Plant Protection: A Biochemical Perspective

Dehydrins are intrinsically disordered proteins composed of several well conserved sequence motifs known as the Y-, S-, F-, and K-segments, the latter of which is a defining feature of all dehydrins. These segments are interspersed by regions of low sequence conservation and are organized modularly, which results in seven different architectures: Kn, SKn, YnSKn, YnKn, KnS, FnK and FnSKn. Dehydrins are expressed ubiquitously throughout the plant kingdom during periods of low intracellular water content, and are capable of improving desiccation tolerance in plants. In vitro evidence of dehydrins shows that they are involved in the protection of membranes, proteins and DNA from abiotic stresses. However, the molecular mechanisms by which these actions are achieved are as of yet somewhat unclear. With regards to macromolecule cryoprotection, there is evidence to suggest that a molecular shield-like protective effect is primarily influenced by the hydrodynamic radius of the dehydrin and to a lesser extent by the charge and hydrophobicity. The interaction between dehydrins and membranes is thought to be a surface-level, charge-based interaction that may help to lower the transition temperature, allowing membranes to maintain fluidity at low temperatures and preventing membrane fusion. In addition, dehydrins are able to protect DNA from damage, showing that these abiotic stress protection proteins have multiple roles

Conserved sequence motifs in the abiotic stress response protein late embryogenesis abundant 3.

LEA3 proteins, a family of abiotic stress proteins, are defined by the presence of a tryptophan-containing motif, which we name the W-motif. We use Pfam LEA3 sequences to search the Phytozome database to create a W-motif definition and a LEA3 sequence dataset. A comprehensive analysis of these sequences revealed four N-terminal motifs, as well as two previously undiscovered C-terminal motifs that contain conserved acidic and hydrophobic residues. The general architecture of the LEA3 sequences consisted of an N-terminal motif with a potential mitochondrial transport signal and the twin-arginine motif cut-site, followed by a W-motif and often a C-terminal motif. Analysis of species distribution of the motifs showed that one architecture was found exclusively in Commelinids, while two were distributed fairly evenly over all species. The physiochemical properties of the different architectures showed clustering in a relatively narrow range compared to the previously studied dehydrins. The evolutionary analysis revealed that the different sequences grouped into clades based on architecture, and that there appear to be at least two distinct groups of LEA3 proteins based on their architectures and physiochemical properties. The presence of LEA3 proteins in non-vascular plants but their absence in algae suggests that LEA3 may have arisen in the evolution of land plants

Disorder and function: a review of the dehydrin protein family

Dehydration proteins (dehydrins) are group 2 members of the late embryogenesis abundant (LEA) protein family. The protein architecture of dehydrins can be described by the presence of three types of conserved sequence motifs that have been named the K-, Y- and S-segments. By definition, a dehydrin must contain at least one copy of the lysine-rich K-segment. Abiotic stresses such as drought, cold, and salinity cause the upregulation of dehydrin mRNA and protein levels. Despite the large body of genetic and protein evidence of the importance of these proteins in stress response, the in vivo protective mechanism is not fully known. In vitro experimental evidence from biochemical assays and localization experiments suggest multiple roles for dehydrins, including membrane protection, cryoprotection of enzymes, and protection from reactive oxygen species. Membrane binding by dehydrins is likely to be as a peripheral membrane protein, since the protein sequences are highly hydrophilic and contain many charged amino acids. Because of this, dehydrins in solution are intrinsically disordered proteins, that is, they have no well-defined secondary or tertiary structure. Despite their disorder, dehydrins have been shown to gain structure when bound to ligands such as membranes, and to possibly change their oligomeric state when bound to ions. We review what is currently known about dehydrin sequences and their structures, and examine the various ligands that have been shown to bind to this family of proteins

Large university classes: A 100+ year-old problem

We think that large university classes are a relatively recent problem, but the literature shows that it has existed for over 100 years. There is a growing body of research on class size and how it relates to student achievement however, the findings have been complex, difficult to interpret, and challenging to integrate. The primary complication is that the definition of a large class is fluid. It can vary with discipline, year level, format of class, and opinion. Because of the complexity, researchers often quantify it, somewhat arbitrarily, at greater than 100 students. One study defines a large class as one that necessitates a change of teaching methods, but also notes that pedagogical approach depends on the class size, creating a circular argument. Irrespective of the problem, we should be able to detect when a change of teaching method was implemented as a course grew. Today, course with enrollments close to 1000 students are common; courses that are often prerequisites for several programs of study. It is therefore becoming more critical to provide educators with valid, reliable, and instructive information on how to effectively teach a large class, and to enable administrators to evaluate and change their practices. In this session, we will discuss the results of a meta-analysis of large classes we performed to define the size of a large class, determine what has driven changes in class size other than costs, and to see if there is an optimal class size that balances competing administration and pedagogical needs

ABBREVIATIONS: AA: INFLAMMATION ASSOCIATED AMYLOIDOSIS,

Graether et al

Freezing of a Fish Antifreeze Protein Results in Amyloid Fibril Formation

Amyloid is associated with a number of diseases including Alzheimer's, Huntington's, Parkinson's, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the α-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils