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1.  Atomic Force Fluorescence Microscopy in the Characterization of Amyloid Fibril Assembly and Oligomeric Intermediates 
Atomic force microscopy (AFM) has become a conventional tool for elucidation of the molecular mechanisms of protein aggregation and, specifically, for analysis of assembly pathways, architecture, aggregation state, and heterogencity of oligomeric intermediates or mature fibrils. AFM imaging provides useful information about particle dimensions, shape, and substructure with nanometer resolution. Conventional AFM methods have been very helpful in the analysis of polymorphic assemblies formed in vitro from homogeneous proteins or peptides. However, AFM imaging on its own provides limited insight into conformation or composition of assemblies produced in the complex environment of a cell, or prepared from a mixture of proteins as a result of cross-seeding. In these cases, its combination with fluorescence microscopy (AFFM) increases its resolution.
doi:10.1007/978-1-61779-551-0_11
PMCID: PMC3786444  PMID: 22528089
Amyloids; Assembly; Atomic force microscopy; Atomic force fluorescence microscopy; Immunofluorescence; Oligomers
2.  Dynamic Diagnosis of Familial Prion Diseases Supports the β2-α2 Loop as a Universal Interference Target 
PLoS ONE  2011;6(4):e19093.
Background
Mutations in the cellular prion protein associated to familial prion disorders severely increase the likelihood of its misfolding into pathogenic conformers. Despite their postulation as incompatible elements with the native fold, these mutations rarely modify the native state structure. However they variably have impact on the thermodynamic stability and metabolism of PrPC and on the properties of PrPSc aggregates. To investigate whether the pathogenic mutations affect the dynamic properties of the HuPrP(125-229) α-fold and find possible common patterns of effects that could help in prophylaxis we performed a dynamic diagnosis of ten point substitutions.
Methodology/Principal Findings
Using all-atom molecular dynamics simulations and novel analytical tools we have explored the effect of D178N, V180I, T183A, T188K, E196K, F198S, E200K, R208H, V210I and E211Q mutations on the dynamics of HuPrP(125-228) α-fold. We have found that while preserving the native state, all mutations produce dynamic changes which perturb the coordination of the α2-α3 hairpin to the rest of the molecule and cause the reorganization of the patches for intermolecular recognition, as the disappearance of those for conversion inhibitors and the emergence of an interaction site at the β2-α2 loop region.
Conclusions/Significance
Our results suggest that pathogenic mutations share a common pattern of dynamical alterations that converge to the conversion of the β2-α2 loop into an interacting region that can be used as target for interference treatments in genetic diseases.
doi:10.1371/journal.pone.0019093
PMCID: PMC3084259  PMID: 21552571
3.  Detection of oxidized methionine in selected proteins, cellular extracts, and blood serums by novel anti-methionine sulfoxide antibodies 
Methionine sulfoxide (MetO) is a common posttranslational modification to proteins occurring in vivo. These modifications are prevalent when reactive oxygen species levels are increased. To enable the detection of MetO in pure and extracted proteins from various sources, we have developed novel antibodies that can recognize MetO-proteins. These antibodies are polyclonal antibodies raised against an oxidized methionine-rich zein protein (MetO-DZS18) that are shown to recognize methionine oxidation in pure proteins and mouse and yeast extracts. Furthermore, mouse serum albumin and immunoglobulin (IgG) were shown to accumulate MetO as function of age especially in serums of methionine sulfoxide reductase A knockout mice. Interestingly, high levels of methionine-oxidized IgG in serums of subjects diagnosed with Alzheimer’s disease were detected by western blot analysis using these antibodies. It is suggested that anti-MetO-DZS18 antibodies can be applied in the identification of proteins that undergo methionine oxidation under oxidative stress, aging, or disease state conditions.
doi:10.1016/j.abb.2009.01.020
PMCID: PMC2768404  PMID: 19388147
Oxidative stress; post-translation modification; aging; neurodegenerative diseases; Alzheimer’s disease; prion protein; serum proteins

Results 1-3 (3)