The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSDlocalized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission. Neurons are highly polarized cells, specializing in the reception of numerous, independent signal inputs and rapid integration of these inputs into an electrochemical response. The major sites of signal input are synapses, which are highly ordered cell junctions formed between two neurons and are typically unidirectional in fast excitatory chemical neurotransmission in the mammalian CNS. The response to neurotransmitter (NT) 1 release at the synapse is provided by a protein matrix of NT receptors and supporting proteins collectively known as the postsynaptic density (PSD) (for review, see Refs. 1-3). The PSD has several proposed functions including: signal amplification, cytoskeletal anchorage, biochemical signaling regulation, and NT receptor clustering (1, 4 -6).Changes in size and composition of the PSD correlate with changes in synaptic strength (7,8), including alterations that are stably maintained such as long-term potentiation (LTP), a physiologically relevant increase in synaptic efficacy and a model for learning and memory (9, 10). Therefore, an understanding of the protein composition of the PSD is a prerequisite for modeling the molecular interactions regulating synaptic strength.The structure of PSDs purified from rodent brains using gradient centrifugation and Triton X-100 extraction has been shown by electron microscopy (EM) to be virtually identical to the "in vivo" PSD structure (4, 11). Gel electrophoresis, enzymatic activity assays, and EM experiments have demonstrated that this procedure yields a highly pure, membranefree PSD fraction (11,12). Recent proteomic studies have investigated the composition of the PSD by SDS-PAGE or two-dimensional gel electrophoresis (2DE) coupled with MS (13-16). Li et al. (16) also performed shotgun proteomics using cysteine-containing peptides selected using ICAT techniques. However, each of these investigations identified less than one-third of previously described and biochemically confirmed PSD components, pointing to limitations in the techniques used. A recent paper by Yoshimura et al. (17) reports the identification by mass spectrometry of 492 proteins in the PSD, which suggests that the PSD is more ...

Experimental and theoretical evidence is provided that indicates the presence of inclusion complexes in the gas phase when cyclodextrin and amino acid mixtures are electrosprayed into a Fourier transform mass spectrometer. A guest exchange reaction that is enantiospecific is used to probe the structure of the gas-phase complex. Chiral selectivity is affected by both the size of the guest and the size of the cavity. These observations are based on a selected number of amino acids with various hosts. The experimental results are supported by molecular dynamics calculations. We further conclude that rather than nonspecific complexes, amino acidcyclodextrin complexes produced in solution maintain the included structure even in the gas phase.

The RZZ complex recruits dynein to kinetochores. We investigated structure, topology, and interactions of the RZZ subunits (ROD, ZWILCH, and ZW10) in vitro, in vivo, and in silico. We identify neuroblastoma-amplified gene (NAG), a ZW10 binder, as a ROD homolog. ROD and NAG contain an N-terminal beta propeller followed by an alpha solenoid, which is the architecture of certain nucleoporins and vesicle coat subunits, suggesting a distant evolutionary relationship. ZW10 binding to ROD and NAG is mutually exclusive. The resulting ZW10 complexes (RZZ and NRZ) respectively contain ZWILCH and RINT1 as additional subunits. The X-ray structure of ZWILCH, the first for an RZZ subunit, reveals a novel fold distinct from RINT1's. The evolutionarily conserved NRZ likely acts as a tethering complex for retrograde trafficking of COPI vesicles from the Golgi to the endoplasmic reticulum. The RZZ, limited to metazoans, probably evolved from the NRZ, exploiting the dynein-binding capacity of ZW10 to direct dynein to kinetochores.

The validity of the "three-point interaction" model is examined in the guest exchange reaction involving complexes of cyclodextrins and amino acids. The amino acid guest is exchanged in the gas phase in the presence of a gaseous alkyl amine. The net reaction is proton transfer between the protonated amino acid and the alkyl amine. The amino acid is lost as a neutral species. This reaction is sensitive to the chirality of the amino acid. Several amino acids are examined as well as the respective methyl esters to determine the role of the three interacting groups (ammonium, carboxylic acid, and side chain) in enantioselectivity. We find that the three-point interaction model is indeed valid in the gas phase. Enantioselectivity is optimal when two points of attraction and one repulsion is present in the gas-phase complex. The results are supported by molecular modeling calculations. A mechanism for the exchange is proposed.