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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(More)
Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (P(i)), both forward and backward steps become less load-dependent. From(More)
Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils(More)
The ATPase cycle of GroE chaperonins has been examined by transient kinetics to dissect partial reactions in complexes where GroEL is asymmetrically loaded with nucleotides. The occupation of one heptameric ring by ADP does not inhibit the loading of the other with ATP nor does it prevent the consequent structural rearrangement to the "open" state. However,(More)
Single-molecule approaches permit an unrivalled view of how complex systems operate and have recently been used to understand DNA-protein interactions. These tools have enabled advances in a particularly challenging problem, the search for damaged sites on DNA. DNA repair proteins are present at the level of just a few hundred copies in bacterial cells to(More)
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(More)
The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with(More)
The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were(More)
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(More)
Striated muscle contraction is governed by the thin filament regulatory proteins troponin and tropomyosin. Here, we investigate the molecular mechanisms by which troponin-tropomyosin inhibits myosin's interactions with the thin filament in the absence of calcium by using a laser trap. The displacement events for a single-myosin molecule interacting with a(More)