We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to È2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method-termed photoactivated localization microscopy-to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Increasing evidence suggests that formation and propagation of misfolded aggregates of 42-residue human amyloid β (Aβ(1–42)), rather than the more abundant Aβ(1–40), provokes the Alzheimer’s cascade. To date, structural details of misfolded Aβ(1–42) have remained elusive. Here we present the atomic model of Aβ(1–42) amyloid fibril based on solid-state NMR (SSNMR) data. It displays triple parallel-β-sheet segments that are different from reported structures of Aβ(1–40) fibrils. Remarkably, Aβ(1–40) is not compatible with the triple-β motif, as seeding with Aβ(1–42) fibrils does not promote conversion of monomeric Aβ(1–40) into fibrils via cross-replication. SSNMR experiments suggest that the Ala42 carboxyl terminus, absent in Aβ(1–40), forms a salt-bridge with Lys28 as a self-recognition molecular switch that excludes Aβ(1–40). The results provide insight into Aβ(1–42)-selective self-replicating amyloid propagation machinery in early-stage Alzheimer’s disease.

Cu2+ binding to Alzheimer’s β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer’s senile plaque and such association of Aβ with Cu2+ triggers the production of neurotoxic reactive oxygen species (ROS) such as H2O2. However, detailed binding sites and binding structures of Cu2+ to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu2+ binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution 13C SSNMR for full-length 40-residue Aβ(1–40). Selective quenching observed in 13C SSNMR of Cu2+-bound Aβ(1–40) suggested that primary Cu2+ binding sites in Aβ(1–40) fibrils include Nε in His-13 and His-14, and carboxyl groups in Val-40 as well as in Glu side chains (Glu-3, Glu-11, and/or Glu-22). 13C chemical shift analysis demonstrated no major structural changes upon Cu2+ binding in the hydrophobic core regions (residues 18–25 and 30–36). Although the ROS production via oxidization of Met-35 in the presence of Cu2+ has been long suspected, our SSNMR analysis of 13CεH3-S- in M35 showed little changes after Cu2+ binding, excluding the possibility of Met-35 oxidization by Cu2+ alone. Preliminary molecular dynamics (MD) simulations on Cu2+-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu2+-binding modes unique in Aβ fibril, which are realized by both intra- and inter-molecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.

Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation methods. Studies of a ternary complex formed from the substrate analog folate and oxidized NADP ؉ cofactor revealed conformational exchange between a ground state, in which the active site loops adopt a closed conformation, and a weakly populated (4.2% at 30°C) excited state with the loops in the occluded conformation. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.enzyme catalysis ͉ hydride transfer ͉ NMR relaxation P roteins are dynamic molecular machines, and conformational fluctuations on a wide range of time scales are intimately associated with protein function. It has long been recognized that dynamic processes play an important role in the catalytic function of enzymes (1, 2). Protein motion is implicated in events such as binding of substrate or cofactor, allosteric regulation, and product release, and the catalyzed reaction itself is inherently dynamic, with changes in atomic positions occurring along the reaction coordinate (3). Despite mounting experimental evidence that the active sites of enzymes are inherently flexible (4-6), a detailed understanding of the relationship between the dynamics and thermodynamics of protein fluctuations and the catalytic process is currently lacking.A number of experimental and theoretical investigations have suggested that protein motions play an important role in catalysis by the enzyme dihydrofolate reductase (DHFR) (see refs. 7 and 8 for recent reviews). DHFR catalyzes the reduction of 7,8-dihydrofolate (DHF) through stereo-specific hydride transfer from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor. The enzyme is essential for tetrahydrofolate (THF) biosynthesis, plays a central role in promoting cell growth and proliferation, and is the target of several anticancer and antibiotic drugs. Escherichia coli DHFR has been subjected to extensive kinetic and structural studies that have defined the complete kinetic mechanism (9) and the structural transitions involved in the catalytic cycle (10, 11). During the reaction cycle (Fig. 1), the enzyme progresses through conformations in which the active site loops are in a closed state, in the holoenzyme and the Michaelis complex, and a series of product complexes in which the loops adopt an occluded conformation. In the closed state, the Met-20 loop (residues 9-24) packs against the nicotinamide ring of the cofactor and seals the active site (10). In the occluded state, Met-16 and Glu-17 in the Met-20 loop project into the active site and sterically occlude the binding site for the nicotinamide-r...

Enzyme catalysis can be described as progress over a multidimensional energy landscape where ensembles of interconverting conformational substates channel the enzyme through its catalytic cycle. We applied NMR relaxation dispersion to investigate the role of bound ligands in modulating the dynamics and energy landscape of Escherichia coli dihydrofolate reductase to obtain insights into the mechanism by which the enzyme efficiently samples functional conformations as it traverses its reaction pathway. Although the structural differences between the occluded substrate binary complexes and product ternary complexes are very small, there are substantial differences in protein dynamics. Backbone fluctuations on the μs-ms timescale in the cofactor binding cleft are similar for the substrate and product binary complexes, but fluctuations on this timescale in the active site loops are observed only for complexes with substrate or substrate analog and are not observed for the binary product complex. The dynamics in the substrate and product binary complexes are governed by quite different kinetic and thermodynamic parameters. Analogous dynamic differences in the E:THF:NADPH and E:THF:NADP þ product ternary complexes are difficult to rationalize from ground-state structures. For both of these complexes, the nicotinamide ring resides outside the active site pocket in the ground state. However, they differ in the structure, energetics, and dynamics of accessible higher energy substates where the nicotinamide ring transiently occupies the active site. Overall, our results suggest that dynamics in dihydrofolate reductase are exquisitely "tuned" for every intermediate in the catalytic cycle; structural fluctuations efficiently channel the enzyme through functionally relevant conformational space. catalysis | energy landscape | enzyme dynamics | NMR | relaxation