Technical Reports

NMR relaxation phenomena give insight into local protein motions, and understanding protein dynamics can aid in understanding protein function. Nuclear Overhauser enhancements (NOEs) have been traditionally used to determine protein structure by NMR, but a novel application using exact NOEs provides a structural ensemble that describes a protein's structure as well as its dynamics.

Tryptophan scanning is now used to characterize transient intermediate states in the folding pathway of a protein. By following the intrinsic fluorescence of tryptophan residues introduced at different solvent-exposed positions of model protein ubiquitin, the authors characterize a late folding intermediate.

NTPases use a metal ion, typically Mg²⁺, coordinated by a conserved serine or threonine residue, to enable phosphate binding and catalysis. Now cysteine substitutions at the switch 1 motif of different kinesins render them able to use Mn²⁺ instead of Mg²⁺, allowing their enzymatic and motor activities to be modulated by the ratio of Mg²⁺ to Mn²⁺.

The combination of an F-box domain with a single-domain antibody that recognizes green fluorescent protein (GFP) now allows controlled depletion of GFP fusions in mammalian cells and in flies. This system, called deGradFP, should be widely useful, as GFP fusions are available for many proteins in model organisms.

The interaction of solvent with protein has been a major unresolved and significant problem for decades. Now, NMR techniques characterize the hydration sites of ubiquitin encapsulated within reverse micelles. This approach reveals a clustering of water molecules with similar residence times, an observation that is generally not accessible by crystallographic analyses.

Crystallization of RNA molecules can pose a challenge, and the U1A RNA binding protein has been used to facilitate the process. Now a different portable RNA sequence and a synthetic Fab are presented as tools for RNA structural determination. In addition to functioning as a crystallization chaperone, the Fab also serves as the search model to provide phase information.

Understanding genomic organization in three dimensions may be one key to understanding gene regulation. Through the combination of an advanced version of chromosome conformation capture technology and an integrated modeling platform, the structure of the α-globin locus in cells where it is silent is compared to that in cells with high α-globin expression.

Even though heterogeneous nuclear ribonucleoprotein C (hnRNP C) is among the most abundant proteins in the nucleus, its role in splicing has remained unresolved. Data obtained using a newly developed individual-nucleotide UV crosslinking and immunoprecipitation (iCLIP) technique, integrated with alternative splicing profiles, indicate how the position of hnRNP particles determines their effect on inclusion of alternative exons.

While the topological analysis of protein complexes by electron microscopy (EM) has developed in recent years, the exact position of single subunits within multisubunit complexes has been difficult. A new clonable peptide tag, which when attached to a target protein can recruit a structurally prominent label that is easily identifiable by EM, may help solve this problem.

Membrane proteins pose a huge challenge for structural analysis, but a new study reports the first NMR structure determination of a detergent-solubilized seven-helical transmembrane (7TM) protein, the phototaxis receptor sensory rhodopsin II. This case study may open the doors to similar solution NMR structures for other 7TM proteins.

SH2 domains are present in many proteins, and designing specific inhibitors has been a challenge. Now a monobody, based on a fibronectin type III domain scaffold, that targets the SH2 domain from Abl kinase is developed, with structural data revealing the basis of its specificity and functional work exploring its effects in vitro and in cells.

GFP fluorescence can be modulated in mammalian cells by binding to single-chain antibodies (nanobodies), selected to make GFP brighter or dimmer; these changes are explained by the crystal structures of the GFP-nanobody complexes. The applications of such nanobodies to monitor protein expression and subcellular localization in real time are explored.

Screening a library of artificial zinc fingers for transcriptional activators in mammalian cells can be laborious. Now a strategy is described that couples the screening to production of retroviral particles that will carry the positive clones, allowing iterative rounds of selection.

The protective antigen (PA) moiety of anthrax toxin exists as a stable prepore, converting into the pore form under low pH to translocate the enzymatic components across the host cell membrane. The PA pore rapidly aggregates in solution, and it is now shown that the chaperone GroEL can stabilize the PA pore, allowing single-particle EM analysis. This method could be useful for other membrane protein complexes.