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Gardezi S.R.,Western Research Institute | Taylor P.,Molecular Structure and Function | Stanley E.F.,Western Research Institute
Channels | Year: 2010

CaV2.2 voltage-gated calcium channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with Ca V2.2 in purified presynaptic terminal membranes. A number of known and new CaV2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-Ca V2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct CaV2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of Ca V2.2 was beyond reasonable doubt. The identification of the long-splice CaV2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested. © 2010 Landes Bioscience.

Willer T.,University of Iowa | Willer T.,Howard Hughes Medical Institute | Lee H.,University of California at Los Angeles | Lommel M.,University of Heidelberg | And 20 more authors.
Nature Genetics | Year: 2012

Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy that is accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of diseases associated with abnormal post-translational processing of α-dystroglycan that share a defect in laminin-binding glycan synthesis. Although mutations in six genes have been identified as causes of WWS, only half of all individuals with the disease can currently be diagnosed on this basis. A cell fusion complementation assay in fibroblasts from undiagnosed individuals with WWS was used to identify five new complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the ISPD gene (encoding isoprenoid synthase domain containing). The pathogenicity of the identified ISPD mutations was shown by complementation of fibroblasts with wild-type ISPD. Finally, we show that recessive mutations in ISPD abolish the initial step in laminin-binding glycan synthesis by disrupting dystroglycan O-mannosylation. This establishes a new mechanism for WWS pathophysiology. © 2012 Nature America, Inc. All rights reserved.

Marsh J.A.,University of Cambridge | Teichmann S.A.,University of Cambridge | Forman-Kay J.D.,Molecular Structure and Function | Forman-Kay J.D.,University of Toronto
Current Opinion in Structural Biology | Year: 2012

Protein flexibility spans a broad spectrum, from highly stable folded to intrinsically disordered states. In this review, we discuss how various techniques, including X-ray crystallography, nuclear magnetic resonance spectroscopy and ensemble-modeling strategies employing various experimental measurements, have enabled detailed structural and dynamic characterizations of proteins in their free and bound states. This has revealed a variety of possible binding scenarios in which flexibility can either decrease or increase upon binding. Furthermore, dynamic free-state ensembles have repeatedly been observed to contain transiently formed conformations that partially or completely resemble bound states. These results demonstrate an intimate connection between protein flexibility and protein interactions and illustrate the huge diversity of structure and dynamics in both free proteins and protein complexes. © 2012 Elsevier Ltd.

Forman-Kay J.D.,Molecular Structure and Function | Forman-Kay J.D.,University of Toronto | Mittag T.,St Jude Childrens Research Hospital
Structure | Year: 2013

Intrinsically disordered proteins (IDPs), which lack persistent structure, are a challenge to structural biology due to the inapplicability of standard methods for characterization of folded proteins as well as their deviation from the dominant structure/function paradigm. Their widespread presence and involvement in biological function, however, has spurred the growing acceptance of the importance of IDPs and the development of new tools for studying their structure, dynamics, and function. The interplay of folded and disordered domains or regions for function and the existence of a continuum of protein states with respect to conformational energetics, motional timescales, and compactness are shaping a unified understanding of structure-dynamics-disorder/ function relationships. In the 20th anniversary of Structure, we provide a historical perspective on the investigation of IDPs and summarize the sequence features and physical forces that underlie their unique structural, functional, and evolutionary properties. © 2013 Elsevier Ltd.

Farber P.J.,Molecular Structure and Function | Mittermaier A.,McGill University
Biophysical Reviews | Year: 2015

Allosteric transmission of information between distant sites in biological macromolecules often involves collective transitions between active and inactive conformations. Nuclear magnetic resonance (NMR) spectroscopy can yield detailed information on these dynamics. In particular, relaxation dispersion techniques provide structural, dynamic, and mechanistic information on conformational transitions occurring on the millisecond to microsecond timescales. In this review, we provide an overview of the theory and analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments and briefly describe their application to the study of allosteric dynamics in the homeodomain from the PBX transcription factor (PBX-HD). CPMG NMR data show that local folding (helix/coil) transitions in one part of PBX-HD help to communicate information between two distant binding sites. Furthermore, the combination of CPMG and other spin relaxation data show that this region can also undergo local misfolding, reminiscent of conformational ensemble models of allostery. © 2015, International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag Berlin Heidelberg.

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