Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.

SUMMARY How DNA repair proteins sort through a genome for damage is one of the fundamental unanswered questions in this field. To address this problem, we uniquely labeled bacterial UvrA and UvrB with differently colored quantum dots and visualized how they interacted with DNA individually or together using oblique-angle fluorescence microscopy. UvrA was observed to utilize a three-dimensional search mechanism, binding transiently to the DNA for short periods (7 s). UvrA also was observed jumping from one DNA molecule to another over ~1 μm distances. Two UvrBs can bind to a UvrA dimer and collapse the search dimensionality of UvrA from three to one dimension by inducing a substantial number of UvrAB complexes to slide along the DNA. Three types of sliding motion were characterized: random diffusion, paused motion, and directed motion. This UvrB-induced change in mode of searching permits more rapid and efficient scanning of the genome for damage.

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Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix–hairpin–helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained λ-DNA molecules suspended between 5 µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe111 insertion.

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Background: How calcium regulates thin filament activation is uncertain.Results: Single molecule imaging is used to report on thin filament activation across relevant solution conditions.Conclusion: Two myosin heads are required to activate a thin filament, which occurs as three states, and myosin binding opens up 11 sites for subsequent binders.Significance: This first direct visualization of cooperativity in action exposes how complex systems function.

SUMMARY Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms nonmotile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the β-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of β-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance.