Mapping the amino acid properties of constituent nucleoporins onto the yeast nuclear pore complex

Visualization of molecular structures aids in the understanding of structural and functional roles of biological macromolecules. Macromolecular transport between the cell nucleus and cytoplasm is facilitated by the nuclear pore complex (NPC). The ring structure of the NPC is large and contains several distinct proteins (nucleoporins) which function as a selective gate for the passage of certain molecules into and out of the nucleus. In this note we demonstrate the utility of a python code that allows direct mapping of the physiochemical properties of the constituent nucleoporins on the scaffold of the yeast NPC׳s cytoplasmic view. We expect this tool to be useful for researchers to visualize the NPC based on their physiochemical properties and how it alters when specific mutations are introduced in one or more of the nucleoporins. The code developed using Python is available freely from the authors.     

Microtubules associated with nuclear pore complexes and coated pits in the CNS

Summary Using a new albumin prefixation technique, microtubules have been observed in close association with the nuclear pores of neurons and glia. Thus, microtubules may be involved in such phenomena as anchoring, migration or rotation of the nucleus or in chemical messenger transport between nucleus and cytoplasm. Microtubules are also seen running close to the coated pits of dendrites. The implications are discussed.The authors gratefully acknowledge the support of grants from the British Medical Research Council (E.G.G.) and USPHS grants NS 09678 and NS 04053; National Institutes of Neurological and Communicative Disorders and Stroke (L.E.W.). We thank Hilary Samson for excellent technical collaboration, Julie Barron and Trevor King for technical assistance and Ann Harris for secretarial help     

Perturbing the DNA sequence selectivity of metallointercalator-peptide conjugates by single amino acid modification.

Metallointercalator-peptide conjugates that provide small molecular mimics to explore peptide-nucleic acid recognition have been prepared. Specifically, a family of peptide conjugates of [Rh(phi)(2)(phen')](3+) [where phi = 9,10-phenanthrenequinone diimine and phen' = 5-(amidoglutaryl)-1,10-phenanthroline] has been synthesized and their DNA-binding characteristics examined. Single amino acid modifications were made from the parent metallointercalator-peptide conjugate [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2), which targets 5'-CCA-3' site-specifically. Moving the glutamate at position 10 in the sequence of the appended peptide to position 6 {[Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2)} changed the sequence preference of the metallointercalator-peptide conjugate to 5'-ACA-3'. Subsequent mutation of the glutamate at position 6 to arginine {[Rh(phi)(2)(phen')](3+)-AANVARAAWARAA-CONH(2)} caused more complex changes in DNA recognition. Thermodynamic dissociation constants were determined for these metallointercalator-peptide conjugates by photoactivated DNA cleavage assays with the rhodium intercalators. At 55 degrees C in the presence of 5 mM MnCl(2), [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2) binds to a 5'-CCA-3' site with K(d) = 5.7 x 10(-)(8) M, whereas [Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2) binds to its target 5'-ACA-3' site with K(d) = 9.9 x 10(-8) M. The dissociation constant for [Rh(phi)(2)(phen')](3+) with random-sequence DNA is 7.0 x 10(-7) M. Structural models have been developed and refined to account for the observed sequence specificities. As with much larger DNA-binding proteins, with these metal-peptide conjugate mimics, single amino acid changes can lead to single or multiple base changes in the DNA site targeted.     

Spectroscopic characterization of amino acid and amino acid ester-Schiff-base complexes of oxovanadium and their catalysis in sulfide oxidation

α-Amino acid Schiff-base complexes of oxovanadium(IV), whose ligands have amino acid side chains with coordinating functional groups, retained coordination geometries in which the amino acid side chains were probably coordinated in the axial position with a phenolate oxygen, a carboxylate oxygen, an imine nitrogen, and a solvent being bound in the equatorial plane. As for amino acid ester Schiff-base complexes, the amino acid side chains were coordinated in the equatorial plane in the place of the carboxyl group in the case of the amino acid Schiff-base complexes. The amino acid Schiff-base complexes of oxovanadium(V) were present as dimers in dichloromethane. Peroxo complexes prepared from the Schiff-base complexes of oxovanadium(V) converted methyl phenyl sulfide to the corresponding sulfoxide in 80-90% yield in CDCl₃ and in 30-70% yield in CD₃OD in 30 min. They converted the sulfide in a stereoselective manner yielding the sulfoxide in small enantiomeric excess (5-20%).     

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.     

Kojic acid–amino acid amide metal complexes and their melanogenesis inhibitory activities

Tyrosinase plays a critical role in the early stages of the melanin synthetic pathway by catalyzing the oxidation of the substrate. Therefore, tyrosinase inhibitors have been intensively studied in both cosmetic and food industries to develop hypopigmentary agents and prevent enzymatic browning in food. Previously, we reported that kojic acid–amino acid amide (KA‐AA‐NH₂) showed enhanced tyrosinase inhibitory activity compared with kojic acid alone, but this was not observed in a cell test because of poor cell permeability. To enhance cell permeability, we prepared copper and zinc complexes of KA‐AA‐NH₂ and characterized them using FT‐IR spectroscopy, ESI‐MS spectrometry, and inductively coupled plasma analysis. We then showed that KA‐AA‐NH₂ copper complexes exhibited melanogenesis inhibitory activity in Mel‐Ab cells. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of mismatch repair protein complexes in HeLa nuclear extracts and their interaction with heteroduplex DNA

Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.     

Protein location prediction using atomic composition and global features of the amino acid sequence

Subcellular location of protein is constructive information in determining its function, screening for drug candidates, vaccine design, annotation of gene products and in selecting relevant proteins for further studies. Computational prediction of subcellular localization deals with predicting the location of a protein from its amino acid sequence. For a computational localization prediction method to be more accurate, it should exploit all possible relevant biological features that contribute to the subcellular localization. In this work, we extracted the biological features from the full length protein sequence to incorporate more biological information. A new biological feature, distribution of atomic composition is effectively used with, multiple physiochemical properties, amino acid composition, three part amino acid composition, and sequence similarity for predicting the subcellular location of the protein. Support Vector Machines are designed for four modules and prediction is made by a weighted voting system. Our system makes prediction with an accuracy of 100, 82.47, 88.81 for self-consistency test, jackknife test and independent data test respectively. Our results provide evidence that the prediction based on the biological features derived from the full length amino acid sequence gives better accuracy than those derived from N-terminal alone. Considering the features as a distribution within the entire sequence will bring out underlying property distribution to a greater detail to enhance the prediction accuracy.     

Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs.

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAs and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequences of the bovine subunit.     

Dynamics of the nuclear envelope and of nuclear pore complexes during mitosis in the Drosophila embryo

Early embryonic development in Drosophila melanogaster is marked by a series of thirteen very rapid (10-15 min) and highly synchronous nuclear divisions, the last four of which occur just beneath the embryo surface. A total of some 6000 blastoderm nuclei result, which are subsequently enclosed by furrow membranes to form the cellular blastoderm. We have examined the fine structure of nuclear division in late syncytial embryos. The mitotic spindle forms adjacent to the nuclear envelope on the side facing the embryo surface. During prophase, astral microtubules deform the nuclear envelope which then ruptures at the poles at the onset of prometaphase. The nuclear envelope remains essentially intact elsewhere throughout mitosis. A second envelope begins to form around the nuclear envelope in prometaphase and is completed by metaphase; the entire double layered structure, referred to as the spindle envelope, persists through early in the ensuing interphase. Pole cell spindles are enclosed by identical spindle envelopes. Interphase and prophase nuclei contain nuclear pore complexes (PCs) of standard dimensions and morphology. In prometaphase PCs become much less electron-dense, although they retain their former size and shape. By metaphase, no semblance of PC structure remains, and instead, both layers of the spindle envelope are interrupted by numerous irregular fenestrae. PCs are presumably disassembled into their component parts during mitosis, and reassembled subsequently. Yolk nuclei remain among the central yolk mass when most nuclei migrate to the surface, cease to divide, yet become polyploid. These nuclei nonetheless lose and regain PCs in synchrony with the dividing blastoderm nuclei. In addition, they gain and lose a second fenestrated membrane layer with the same timing. Cytoplasmic membranes containing PCs (annulate lamellae) also lose and regain pores in synchrony with the two classes of nuclear envelopes. The factors that affect the integrity of PCs in dividing blastoderm nuclei appear to affect those in other membrane systems to an equivalent degree and with identical timing.     

smFRET studies of the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding

The selectivity with which a biomolecule can bind its cognate ligand when confronted by the vast array of structurally similar, competing ligands that are present in the cell underlies the fidelity of some of the most fundamental processes in biology. Because they collectively comprise one of only a few methods that can sensitively detect the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding, single-molecule fluorescence, and particularly single-molecule fluorescence resonance energy transfer (smFRET), approaches have revolutionized studies of ligand-binding reactions. Here, we describe a widely used smFRET strategy that enables investigations of a large variety of ligand-binding reactions, and discuss two such reactions, aminoacyl-tRNA selection during translation elongation and splice site selection during spliceosome assembly, that highlight both the successes and challenges of smFRET studies of ligand-binding reactions. We conclude by reviewing a number of emerging experimental and computational approaches that are expanding the capabilities of smFRET approaches for studies of ligand-binding reactions and that promise to reveal the mechanisms that control the selectivity of ligand binding with unprecedented resolution.     

Prediction of the structural class of globular proteins from their amino acid sequence

Two-dimensional representation of consequence of 32 proteins with known three-dimensional structure has been obtained on 20 X 20 matrix of the distribution of amino acid pairs (nearest neighbours). Prediction algorithm of the structural class of globular proteins has been worked out on the basis of the comparison of 20 X 20 matrix of the distribution of amino acid pairs for the proteins of different structural classes. The accuracy of structural class predictions of 32 proteins has been carried out (all the proteins are taken from numerous ones used to obtain the algorithm).     

Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content

The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,6000. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrae origin. Exceptions were the heteroploid mammalian cells which had the same ratio as the diploid mammalian cells despite higher DNA content. The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin maybe the same for at least the vertebrates with low DNA content, At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.     

Prediction of protein motions from amino acid sequence and its application to protein-protein interaction

Background  

Structural flexibility is an important characteristic of proteins because it is often associated with their function. The movement of a polypeptide segment in a protein can be broken down into two types of motions: internal and external ones. The former is deformation of the segment itself, but the latter involves only rotational and translational motions as a rigid body. Normal Model Analysis (NMA) can derive these two motions, but its application remains limited because it necessitates the gathering of complete structural information.     

Demonstration of a DNase-sensitive network associated with the nuclear pore complexes in rat liver nuclei

Rat liver nuclei have been studied by transmission electron microscopy after resuspension in a phosphate-buffered salt solution containing SO²⁻ ₄ as the quantitatively dominant anion. Owing to the high solubility of chromatin in the presence of SO²⁻ ₄ instead of Cl⁻ at isotonicity, nuclei are depleted for chromatin by DNase I digestion in this buffer, eliminating the need for high-salt extraction. This shows that at least 75% of the nuclear pore complexes are associated with fibrogranular structures, which ramify as a network throughout the nucleus, interconnecting the nuclear lamina, interchromatin granule clusters and nucleoli. Perichromatin granules are located in this material proximal to the nuclear pore complexes. Most of the chromatin is removed without major impact on the network, but below a level of 25% residual chromatin there is a considerable reduction of this material, and only about 15% of the connections to the nuclear pore complexes are resistant to digestion with DNase I or streptodornase A and B. The percentage of nuclear pore complexes connected to the network is further reduced by salt extraction and RNase treatment. These results suggest that DNA is an integral part of the network, which presumably plays a role in nucleo-cytoplasmic transport of RNA and protein. Received: 1 September 1998; in revised form: 17 December 1998 / Accepted: 17 December 1998