Screening of ligands for redox-active europium using magnetic resonance imaging.

We report a screening procedure to predict ligand coordination to EuII and EuIII using magnetic resonance imaging in which bright images indicate complexation and dark images indicate no complexation. Here, paramagnetic GdIII is used as a surrogate for EuIII in the screening procedure to enable detection with magnetic resonance imaging. The screening procedure was tested using a set of eight ligands with known coordination to EuII and EuIII, and results were found to be consistent with expected binding. Validation of the screening procedure with known coordination chemistry enables use with new ligands in the future.

Compensatory mutations in the L30e kink-turn RNA-protein complex.

The S. cerevisiae ribosomal protein L30e is an autoregulatory protein that binds to its own pre-mRNA and mature mRNA to inhibit splicing and translation, respectively. The L30e RNA-binding element is a stem-asymmetric loop-stem that forms a kink-turn. A bacterial genetic system was designed to test the ability of protein variants to repress the expression of reporter mRNAs containing the L30e RNA-binding element. Initial screens revealed that changes in several RNA nucleotides had a measurable effect on repression of the reporter by the wild type protein. RNA mutants that reduce repression were screened against libraries of randomly mutagenized L30e proteins. These screens identified a glycine to serine mutation of L30e, which specifically restores activity to an RNA variant containing a U that replaces a helix-capping G. Similarly, an asparagine to alanine mutation was found to suppress a substitution at a position where the L30e RNA nucleotide extends out into the protein pocket. In addition, a compensatory RNA mutation within a defective RNA variant was found. The identification of these suppressors provides new insights into the architecture of a functional binding element and its recognition by an important RNA-binding protein.

Inherent protein structural flexibility at the RNA-binding interface of L30e.

The Saccharomyces cerevisiae ribosomal protein L30 autoregulates its own expression by binding to a purine-rich internal loop in its pre-mRNA and mRNA. NMR studies of L30 and its RNA complex showed that both the internal loop of the RNA as well as a region of the protein become substantially more ordered upon binding. A crystal structure of a maltose binding protein (MBP)-L30 fusion protein with two copies in the asymmetric unit has been determined. The flexible RNA-binding region in the L30 copies has two distinct conformations, one resembles the RNA bound form solved by NMR and the other is unique. Structure prediction algorithms also had difficulty accurately predicting this region, which is consistent with conformational flexibility seen in the NMR and X-ray crystallography studies. Inherent conformational flexibility may be a hallmark of regions involved in intermolecular interactions.

Learning about being a scientist from the vitamin B12 structure collaboration.

This historical note reviews the work that resulted in the publication of the structure of Vitamin B12 . The collaborations and resulting friendships that developed during this time made a strong impression on the author and encouraged her to pursue a scientific career.

Clinical correlation and molecular evaluation confirm that the MLH1 p.Arg182Gly (c.544A>G) mutation is pathogenic and causes Lynch syndrome.

Approximately 25 % of mismatch repair (MMR) variants are exonic nucleotide substitutions. Some result in the substitution of one amino acid for another in the protein sequence, so-called missense variants, while others are silent. The interpretation of the effect of missense and silent variants as deleterious or neutral is challenging. Pre-symptomatic testing for clinical use is not recommended for relatives of individuals with variants classified as 'of uncertain significance'. These relatives, including non-carriers, are considered at high-risk as long as the contribution of the variant to disease causation cannot be determined. This results in continuing anxiety, and the application of potentially unnecessary screening and prophylactic interventions. We encountered a large Irish Lynch syndrome kindred that carries the c.544A>G (p.Arg182Gly) alteration in the MLH1 gene and we undertook to study the variant. The clinical significance of the variant remains unresolved in the literature. Data are presented on cancer incidence within five kindreds with the same germline missense variant in the MLH1 MMR gene. Extensive testing of relevant family members in one kindred, a review of the literature, review of online MMR mutation databases and use of in silico phenotype prediction tools were undertaken to study the significance of this variant. Clinical, histological, immunohistochemical and molecular evidence from these families and other independent clinical and scientific evidence indicates that the MLH1 p.Arg182Gly (c.544A>G) change causes Lynch syndrome and supports reclassification of the variant as pathogenic.

Internal loop mutations in the ribosomal protein L30 binding site of the yeast L30 RNA transcript.

Yeast ribosomal protein L30 binds to an asymmetric, purine-rich internal loop in its transcript to repress its own splicing and translation. The protein-bound form of the stem-internal loop-stem RNA is an example of a kink-turn RNA structural motif. Analysis of kink-turn motifs reveals that in (2 + 5) internal loops, the identities of five nucleotides are very important, while the remaining two may be varied. Previous SELEX experiments on the L30 binding site showed an identical pattern of sequence variation with five nucleotides highly conserved and two positions variable. In this work, internal loop residues were mutated and tested for protein binding in vitro and in vivo. The two sheared G-A pairs, which cannot be mutated without severely weakening L30 binding, make sequence specific contacts with other portions of the RNA and L30 protein. In contrast, the lone nucleotide that protrudes into the protein and an unpaired adenosine make no sequence-specific contacts, and may be mutated without compromising L30 binding. The internal loop allows the formation of a very tight bend that brings the two stems together with cross-strand stacking of two adenines and an interhelical ribose contact. Replacement of a ribonucleotide with a deoxynucleotide adjacent to the internal loop weakens protein binding significantly. In the absence of L30, some of the internal loop residues involved in the formation of the kink-turn motif are protected from chemical modification, indicating that some elements of kink-turn structure may form in the free L30 RNA.