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Stem cells (SCs) are functionally defined by their abilities to self-renew and generate differentiated cells. Although much effort has been focused on defining the common characteristics among various types of SCs, the genetic and functional differences between multipotent and pluripotent SCs have garnered less attention. We report a direct genetic and functional comparison of molecularly defined and clonally related populations of neural SCs (NSCs) and embryonic SCs (ESCs), using the Sox2 promoter for isolation of purified populations by fluorescence-activated cell sorting. A stringent expression profile comparison of promoter-defined NSCs and ESCs revealed a striking dissimilarity, and subsequent chimera analyses confirmed the fundamental differences in cellular potency between these populations. This direct comparison elucidates the molecular basis for the functional differences in pluripotent ESCs and multipotent NSCs.

Understanding the relationship between the structural and functional organization of the human brain is one of the most important goals of neuroscience. Individual variability in brain structure means that it is essential to obtain this information from the same subject. To date, this has been almost impossible. Even though noninvasive functional imaging techniques such as functional MRI (fMRI) are now commonplace, there is no complementary noninvasive structural technique. We present an in vivo method of examining the detailed neuroanatomy of any individual, which can then be correlated with that individual's own functional results. This method utilizes high-resolution structural MRI to identify distinct cortical regions based on cortical lamination structure. We demonstrate that the observed MR lamination patterns relate to myeloarchitecture through a correlation of histology with MRI. In vivo high-resolution MRI studies identify striate cortex, as well as visual area V5, in four individuals, as defined by using fMRI. The anatomical identification of a cortical area (V5/MT) outside of striate cortex is a significant advance, proving it possible to identify extra-striate cortical areas and demonstrating that in vivo structural mapping of the human cerebral cortex is possible.

The [beta]/[alpha] barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S. cerevisiae inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the [beta]/[alpha] barrel. In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well. Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position. An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern. Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element. Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently.

[F.sub.1][F.sub.o]-ATP synthase is the enzyme responsible for most of the ATP synthesis in living systems. The catalytic domain [F.sub.1] of the [F.sub.1][F.sub.o] complex, [F.sub.1]-ATPase, has the ability to hydrolyze ATP. A fundamental problem in the development of a detailed mechanism for this enzyme is that it has not been possible to determine experimentally the relation between the ligand binding affinities measured in solution and the different conformations of the catalytic [beta] subunits ([[beta].sub.TP], [[beta].sub.DP],[[beta].sub.E]) observed in the crystal structures of the mitochondrial enzyme, M[F.sub.1]. Using free energy difference simulations for the hydrolysis reaction ATP+[H.sub.2]O [right arrow] ADP+[P.sub.i] in the [[beta].sub.TP] and [[beta].sub.DP] sites and unisite hydrolysis data, we are able to identify [[beta].sub.TP] as the 'tight' ([K.sub.D] = [10.sup.-12] M, M[F.sub.1]) binding site for ATP and [[beta].sub.DP] as the 'loose' site. An energy decomposition analysis demonstrates how certain residues, some of which have been shown to be important in catalysis, modulate the free energy of the hydrolysis reaction in the [[beta].sub.TP] and [[beta].sub.DP] sites, even though their structures are very similar. Combined with the recently published simulations of the rotation cycle of [F.sub.1]-ATPase, the present results make possible a consistent description of the binding change mechanism of [F.sub.1]-ATPase at an atomic level of detail.

Pre-mRNA splicing takes place in a large and highly dynamic complex known as the spliceosome. Here we report the optimization of a maltose-binding protein (MBP) affinity-purification method to isolate functional spliceosomes for electron microscopic analysis. Visualization of the spliceosome preparations revealed distinct 40-60 nm particles. Immunogold-conjugated antibodies to spliceosome components specifically label these particles, which are eliminated by treatment with either RNase or protease. Moreover, spliceosomes assembled on two different pre-mRNAs are indistinguishable. This first visualization of purified functional spliceosomes assembled in vitro reveals striking structural features, including one or more central cavities and multiple elongate lobes.

We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle [Ca.sup.2+] release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41-420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22[Angstrom] structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [[sup.3]H]NA[D.sup.+], supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor.

Atomic force microscopy is an exciting new single-molecule technique to add to the toolbox of protein (un)folding methods. However, detailed analysis of the unfolding of proteins on application of force has, to date, relied on protein molecular dynamics simulations or a qualitative interpretation of mutant data. Here we describe how protein engineering [PHI] value analysis can be adapted to characterize the transition states for mechanical unfolding of proteins. Single-molecule studies also have an advantage over bulk experiments, in that partial [PHI] values arising from partial structure in the transition state can be clearly distinguished from those averaged over alternate pathways. We show that unfolding rate constants derived in the standard way by using Monte Carlo simulations are not reliable because of the errors involved. However, it is possible to circumvent these problems, providing the unfolding mechanism is not changed by mutation, either by a modification of the Monte Carlo procedure or by comparing mutant and wild-type data directly. The applicability of the method is tested on simulated data sets and experimental data for mutants of titin 127.

The conformational changes in Ras that accompany the hydrolysis of GTP are critical to its function as a molecular switch in signaling pathways. Understanding how GTP is hydrolyzed by revealing the sequence of intermediary structures in the reaction is essential for understanding Ras signaling. Until now, no structure of an intermediate in GTP hydrolysis has been experimentally determined for Ras alone. We have solved the crystal structure of the Ala-59 to Gly mutant of Ras, (RasA59G), bound to guanosine 5'-imidotriphosphate or GDP to 1.7-[Angstrom] resolution. In the guanosine 5'-imidotriphosphate-bound form, this mutant adopts a conformation that is intermediate between the GTP- and GDP-bound forms of wild-type Ras and that is similar to what has been predicted by molecular dynamics simulation [Ma, J. P. & Karplus, M. (1997) Proc. Natl. Acad. Sci. USA 94, 11905-11910]. This conformation is stabilized by direct and water-mediated interactions between the switch 1 and switch 2 regions and is characterized by an increase in the binding affinity for GTP. We propose that the structural changes promoted by the Ala-59 to Gly mutation exhibit a discrete conformational state assumed by wild-type Ras during GTP hydrolysis.

7,8-Dihydro-8-oxoguanine (8-oxoG) is the most common form of oxidative DNA damage in human cells. Biochemical studies have shown that 8-oxoG decreases the DNA cleavage activity of human topoisomerase I, an enzyme vital to DNA metabolism and stability. We present the 3.1-[Angstrom] crystal structure of human topoisomerase I in noncovalent complex with a DNA oligonucleotide containing 8-oxoG at the +1 position in the scissile strand. We find that 8-oxoG reorganizes the active site of human topoisomerase I into an inactive conformation relative to the structures of topoisomerase I-DNA complexes elucidated previously. The catalytic Tyr-723-Phe rotates away from the DNA cleavage site and packs into the body of the molecule. A second active-site residue, Arg-590, becomes disordered and is not observed in the structure. The docked, inactive conformation of Tyr-723-Phe is reminiscent of the related tyrosine recombinase family of integrases and recombinases, suggesting a common regulatory mechanism. We propose that human topoisomerase I binds to DNA first in an inactive conformation and then rearranges its active site for catalysis. 8-OxoG appears to impact topoisomerase I by stabilizing the inactive, DNA-bound state.

Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. Here, evidence for interactions among components of the Agrobacterium tumefaciens vir-encoded T4SS is presented. The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions. The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins. We established interactions between VirB11 (an inner membrane pore-forming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component). We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A. tumefaciens envelope and including a potential pore protein. In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS). VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components. The identification of these interactions suggests a model for assembly of the T4SS.

The covalent attachment of ubiquitin is an important determinant for selective protein degradation by the 26S proteasome in plants and animals. The specificity of ubiquitination is often controlled by ubiquitin-protein ligases (or E3s), which facilitate the transfer of ubiquitin to appropriate targets. One ligase type, the SCF E3s are composed of four proteins, cullin1/Cdc53, Rbx1/Roc1/Hrt1, Skp1, and an F-box protein. The F-box protein, which identifies the targets, binds to the Skp1 component of the complex through a degenerate N-terminal [approximately equal to] 60-aa motif called the F-box. Using published F-boxes as queries, we have identified 694 potential F-box genes in Arabidopsis, making this gene superfamily one of the largest currently known in plants. Most of the encoded proteins contain interaction domains C-terminal to the F-box that presumably participate in substrate recognition. The F-box proteins can be classified via a phylogenetic approach into five major families, which can be further organized into multiple subfamilies. Sequence diversity within the subfamilies suggests that many F-box proteins have distinct functions and/or substrates. Representatives of all of the major families interact in yeast two-hybrid experiments with members of the Arabidopsis Skp family supporting their classification as F-box proteins. For some, a limited preference for Skps was observed, suggesting that a hierarchical organization of SCF complexes exists defined by distinct Skp/F-box protein pairs. Collectively, the data shows that Arabidopsis has exploited the SCF complex and the ubiquitin/26S proteasome pathway as a major route for cellular regulation and that a diverse array of SCF targets is likely present in plants.

[Na.sup.+]/[H.sup.+] exchanger regulatory factor (NHERF)-1 and NHERF-2, two structurally related protein adapters containing tandem PSD-95/ Discs large/ZO-1 (PDZ) domains, were identified as essential factors for protein kinase A-mediated inhibition of the sodium-hydrogen exchanger, NHE3. NHERF-1 and NHERF-2 also bound other cellular targets including the sodium-phosphate cotransporter type IIa encoded by the NPT2 gene. Targeted disruption of the mouse NHERF-1 gene eliminated NHERF-1 expression in kidney and other tissues of the mutant mice without altering NHERF-2 levels in these tissues. NHERF-1 (+/-) and (-/-) male mice maintained normal blood electrolytes but showed increased urinary excretion of phosphate when compared with wild-type (+/+) animals. Although the overall levels of renal NHERF-1 targets, NHE3 and Npt2, were unchanged in the mutant mice, immunocytochemistry showed that the Npt2 protein was aberrantly localized at internal sites in the renal proximal tubule cells. The mislocalization of Npt2 paralleled a reduction in the transporter protein in renal brush-border membranes isolated from the mutant mice. In contrast, NHE3 was appropriately localized at the apical surface of proximal tubules in both wild-type and mutant mice. These data suggested that NHERF-1 played a unique role in the apical targeting and/or trafficking of Npt2 in the mammalian kidney, a function not shared by NHERF-2 or other renal PDZ proteins. Phosphate wasting seen in the NHERF-1(-/-) null mice provided a new experimental system for defining the role of PDZ adapters in the hormonal control of ion transport and renal disease.

The structure of the reaction center from Rhodobacter sphaeroides has been solved by using x-ray diffraction at a 2.55-[Angstrom] resolution limit. Three lipid molecules that lie on the surface of the protein are resolved in the electron density maps. In addition to a cardiolipin that has previously been reported [McAuley, K. E., Fyfe, P. K., Ridge, J. P., Isaacs, N. W., Cogdell, R. J. & Jones, M. R. (1999) Proc. Natl. Acad. Sci. USA 96, 14706-14711], two other major lipids of the cell membrane are found, a phosphatidylcholine and a glucosylgalactosyl diacylglycerol. The presence of these three lipids has been confirmed by laser mass spectroscopy. The lipids are located in the hydrophobic region of the protein surface and interact predominately with hydrophobic amino acids, in particular aromatic residues. Although the cardiolipin is over 15 [Angstrom] from the cofactors, the other two lipids are in close contact with the cofactors and may contribute to the difference in energetics for the two branches of cofactors that is primarily responsible for the asymmetry of electron transfer. The glycolipid is 3.5 [Angstrom] from the active bacteriochlorophyll monomer and shields this cofactor from the solvent in contrast to a much greater exposed surface evident for the inactive bacteriochlorophyll monomer. The phosphate atom of phosphatidylcholine is 6.5 [Angstrom] from the inactive bacteriopheophytin, and the associated electrostatic interactions may contribute to electron transfer rates involving this cofactor. Overall, the lipids span a distance of [approximately equal to] 30 [Angstrom], which is consistent with a bilayer-like arrangement suggesting the presence of an 'inner shell' of lipids around membrane proteins that is critical for membrane function.

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-L-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.

Early conformational events during folding of acyl-CoA binding protein (ACBP), an 86-residue [alpha]-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 [micro]s to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence energy transfer. Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant [tau] = 80 [micro]s, indicating formation of an intermediate with only slightly enhanced florescence of Trp-55 and Trp-58 relative to the unfolded state. To amplify this phase, a dansyl fluorophore was introduced at the C terminus by labeling an 186C mutant of ACBP with 5-IAEDANS [5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid]. Continuous-flow refolding of guanidine HCl-denatured ACBP showed a major increase in tryptophan-dansyl fluorescence energy transfer, indicating formation of a partially collapsed ensemble of states on the 100-[micro]s time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native state. The kinetic data are fully accounted for by three-state mechanisms with either on- or off-pathway intermediates. The intermediate accumulates to a maximum population of 40%, and its stability depends only weakly on denaturant concentration, which is consistent with a marginally stable ensemble of partially collapsed states with ~1/3 of the solvent-accessible surface buried. The findings indicate that ultrafast mixing methods combined with sensitive conformational probes can reveal transient accumulation of intermediate states in proteins with apparent two-state folding mechanisms.

Tetrameric hemoglobins are the most widely used systems in studying protein cooperativity. Allosteric effects in hemoglobins arise from the switch between a relaxed (R) state and a tense (T) state occurring upon oxygen release. Here we report the 2.0-[Angstrom] crystal structure of the main hemoglobin component of the Antarctic fish Trematomus newnesi, in a partial hemichrome form. The two [alpha]-subunit iron atoms are bound to a CO molecule, whereas in the [beta] subunits the distal histidine residue is the sixth ligand of the heme iron. This structure, a tetrameric hemoglobin in the hemichrome state, demonstrates that the iron coordination by the distal histidine, usually associated with denaturing states, may be tolerated in a native-like hemoglobin structure. In addition, several features of the tertiary and quaternary organization of this structure are intermediate between the R and T states and agree well with the R [right arrow] T transition state properties obtained by spectroscopic and kinetic techniques. The analysis of this structure provides a detailed pathway of heme-heme communication and it indicates that the plasticity of the [beta] heme pocket plays a role in the R [right arrow] T transition of tetrameric hemoglobins. protein denaturation | protein function | x-ray structure | Antarctic

The mechanism of morphological phase transitions was studied for rod-shaped supramolecular assemblies comprised of a poly(acrylic acid)-block-poly(methyl acrylate)-block-polystyrene (PA[A.sub.90]-b-PM[A.sub.80]-b-P[S.sub.100]) triblock copolymer in 33% tetrahydrofuran/water after perturbation by reaction with a positively charged water-soluble carbodiimide. Tetrahydrofuran solvation of the hydrophobic core domain provided the dynamic nature required for the rod-to-sphere phase transition to be complete within 30 min. The intermediate morphologies such as fragmenting rods and pearl-necklace structures were trapped kinetically by the subsequent addition of a diamino crosslinking agent, which underwent covalent crosslinking of the shell layer. Alternatively, shell-crosslinked rod-shaped nanostructures with preserved morphology were obtained by the addition of the crosslinking agent before the addition of the carbodiimide, which allowed for the shell crosslinking to be performed at a faster rate than the morphological reorganization. The formation of robust shell-crosslinked nanostructures provides a methodology by which the morphological evolution processes can be observed, and it allows access to otherwise thermodynamically unstable nanostructures.

Dendritic microenvironments are analogous to local environments created within protein superstructures. Correspondingly, properties of functional cores such as molecular recognition and catalytic activity are profoundly influenced by the surrounding dendritic branches.

Selenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of [H.sub.2][O.sub.2] and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals.

Results reveal that the reactivity of electron transfer cross-reactions is governed by the inner-shell bond reorganization energy. Data substantiate a structure-reactivity relationships and electron-transfer theory and show agreement with Marcus theory.

Likelihood estimates of local rates of evolution within proteins reveal that selective constraints on structure and function are quantitatively stable over billions of years of divergence. The stability of constraints produces an intramolecular clock that gives each protein a characteristic pattern of evolutionary rates along its sequence. This pattern allows the identification of constrained regions and, because the rate of evolution is a quantitative measure of the strength of the constraint, of their functional importance. We show that results from such analyses, which require only sequence alignments, are consistent with experimental and mutational data. The methodology has significant predictive power and may be used to guide structure-function studies for any protein represented by a modest number of homologs in sequence databases.

To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the [alpha]-cobratoxin-[alpha]7 receptor complex. This model was achieved by using (i) a three-dimensional model of the [alpha]7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the [alpha]-cobratoxin and [alpha]7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. The three-dimensional structure of the toxin-receptor complex model was validated a posteriori by an additional double-mutant experiment. The model shows that the toxin interacts perpendicularly to the receptor axis, in an equatorial position of the extracellular domain. The tip of the toxin central loop plugs into the receptor between two subunits, just below the functional receptor loop C, the C-terminal tail of the toxin making adjacent additional interactions at the receptor surface. The receptor establishes major contacts with the toxin by its loop C, which is assisted by principal (loops A and B) and complementary (loops D, F, and 1) functional regions. This model explains the antagonistic properties of the toxin toward the neuronal receptor and opens the way to the design of new antagonists.

We report here an alternative double-helical structure of the DNA molecule. It has been found in the d(ATA[sup.Br]UAT) and d(ATATAT) sequences by single-crystal x-ray crystallography. This sequence is found not only in TATA boxes, but also in other regulatory regions of DNA. Bases of the two antiparallel strands form Hoogsteen pairs, with adenines in the syn conformation. The structure is related neither to those found in triple helices nor to parallel DNA duplexes. Its conformational parameters are very similar to those of duplex DNA in the B form. Both forms may coexist under physiological conditions, although the Hoogsteen pairing greatly influences the recognition sites on DNA. Our results demonstrate that an alternative to the classical B-DNA double helix is possible.

The three PPAR (peroxisome proliferator-activated receptor) isoforms are critical regulators of lipid homeostasis by controlling the balance between the burning and storage of long chain fatty acids. Whereas PPAR[alpha] and PPAR[gamma] have been studied extensively, the function of PPAR[delta] remains the most elusive. Intriguingly, in cotransfection experiments, PPAR[delta] is a potent inhibitor of ligand-induced transcriptional activity of PPAR[alpha] and PPAR[gamma]. This inhibition is achieved, in part, by binding of PPAR[delta] to a peroxisome proliferator response element and the association of nonliganded PPAR[delta] with corepressors SMRT (silencing mediator for retinoid and thyroid hormone receptors), SHARP (SMRT and histone deacetylase-associated repressor protein), and class I histone deacetylases. Stable expression of PPAR[delta] inhibits the expression of endogenous PPAR[alpha] target gene expression in 3T3-PPAR[alpha] cells, whereas a PPAR[delta] mutant that does not interact with the corepressor SMRT loses its ability to repress the induction of PPAR[alpha] target gene. Similarly, stable expression of PPAR[delta] in 3T3-PPAR[gamma] cells leads to inhibition of PPAR[gamma] target gene expression and PPAR[gamma]-mediated adipogenesis. Given the widespread expression of PPAR[delta] and the restricted pattern for PPAR[alpha] and PPAR[gamma], these results suggest a role for PPAR[delta] as a gateway receptor whose relative levels of expression can be used to modulate PPAR[alpha] and PPAR[gamma] activity.

A method for optimizing potential-energy functions of proteins is proposed. The method assumes a hierarchical structure of the energy landscape, which means that the energy decreases as the number of native-like elements in a structure increases, being lowest for structures from the native family and highest for structures with no native-like element. A level of the hierarchy is defined as a family of structures with the same number of native-like elements (or degree of native likeness). Optimization of a potential-energy function is aimed at achieving such a hierarchical structure of the energy landscape by forcing appropriate free-energy gaps between hierarchy levels to place their energies in ascending order. This procedure is different from methods developed thus far, in which the energy gap and/or the Z score between the native structure and all non-native structures are maximized, regardless of the degree of native likeness of the non-native structures. The advantage of this approach lies in reducing the number of structures with decreasing energy, which should ensure the searchability of the potential. The method was tested on two proteins, PDB ID codes 1FSD and 1IGD, with an off-lattice united-residue force field. For 1FSD, the search of the conformational space with the use of the conformational space annealing method and the newly optimized potential-energy function found the native structure very quickly, as opposed to the potential-energy functions obtained by former optimization methods. After even incomplete optimization, the force field obtained by using 1IGD located the native-like structures of two peptides, 1FSD and betanova (a designed three-stranded [beta]-sheet peptide), as the lowest-energy conformations, whereas for the 46-residue N-terminal fragment of staphylococcal protein A, the native-like conformation was the second-lowest-energy conformation and had an energy 2 kcal/mol above that of the lowest-energy structure.

This article discusses the current status of the chemistry of catalysis of the biotin-dependent enzymes with reference to carboxylases, protein sequence, and structural information.

Results demonstrate that the inhibitory action of microgonotropens on the binding of transcription factors to their DNA binding sites is governed by an increased complexation and conformational changes of double-stranded DNA and enhanced ability of transcription factor-induced inhibition of double-stranded DNA binding.

PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. coli. It is composed of two domains, each of which contains a tryptophan residue (Trp-36 and Trp-128, in the N- and C-terminal domains, respectively). To explore the role of domain--domain interactions during folding, the protein was labeled with 6-fluorotryptophan for use in [sup.19]F-NMR experiments. [sup.19]F-NMR data collected as a function of urea concentration revealed the presence of a resonance caused by Trp-128 that was distinct from either the folded or unfolded resonances. The time course of refolding from urea was monitored by stopped-flow fluorescence, CD, and [sup.19]F-NMR, each method showing multiple kinetic phases. The [sup.19]F-NMR stopped-flow spectra, collected at 70 [micro]M of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process ( flourescence | circular dichroism | folding intermediates | P pili

The interplay between structure-search of the native structure and desolvation in protein folding has been explored using a minimalist model. These results support a folding mechanism where most of the structural formation of the protein is achieved before water is expelled from the hydrophobic core. This view integrates water expulsion effects into the funnel energy landscape theory of protein folding. Comparisons to experimental results are shown for the SH3 protein. After the folding transition, a near-native intermediate with partially solvated hydrophobic core is found. This transition is followed by a final step that cooperatively squeezes out water molecules from the partially hydrated protein core.

This review discusses the research done in the area of AMPA receptor trafficking in long-term potentiation and long-term depression pathways. Results indicate that specific AMPA receptor subunits interact with their cognate proteins in the final ralization of activity-dependent changes in synaptic function and plasticity.

Research employs a variety of nuclear paramagnetic resonance pulse sequences for elucidating multistranded DNA architectures containing hydrogen-bonded networks. Results indicate the presence of hydrogen bonds in two quadruplex DNA structures exhibiting triad, tetrad, and hexad motifs involving Watson-Crick, G-G and sheared G.A mismatch base pairing.

The crystal structure of the heterotrimeric quinohemoprotein amine dehydrogenase from Paracoccus denitrificans has been determined at 2.05-[Angstrom] resolution. Within an 82-residue subunit is contained an unusual redox cofactor, cysteine tryptophylquinone (CTQ), consisting of an orthoquinone-modified tryptophan side chain covalently linked to a nearby cysteine side chain. The subunit is surrounded on three sides by a 489-residue, four-domain subunit that includes a diheme cytochrome c. Both subunits sit on the surface of a third subunit, a 337-residue seven-bladed [beta]-propeller that forms part of the enzyme active site. The small catalytic subunit is internally crosslinked by three highly unusual covalent cysteine to aspartic or glutamic acid thioether linkages in addition to the cofactor crossbridge. The catalytic function of the enzyme as well as the biosynthesis of the unusual catalytic subunit is discussed.

The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/ 24p [much greater than] Sec13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated vesicles. Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31. Partially uncoated profiles revealed strands of Sec13/31p stripped from the vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat. coat proteins | liposome | endoplasmic reticulum membrane | transport vesicle

Four-stranded guanine tetraplexes in RNA have been identified to be involved in crucial biological functions, such as dimerization of retroviral RNA, translational repression, and mRNA turnover. However, the structural basis for these biological processes is still largely unknown. Here we report the RNA tetraplex structure [(UGGGGU).sub.4] at ultra-high resolution (0.61 [Angstrom]). The space group is P4[2.sub.1]2, and cell constants are a = b = 36.16 [Angstrom] and c = 74.09 [Angstrom]. The structure was solved by the multiple-wavelength anomalous dispersion method using a set of three-wavelength data of the isomorphous bromo derivative [sup.br]UGGGGU and refined to 0.61-[Angstrom] resolution. Each of the four strands in the asymmetric unit forms a parallel tetraplex with symmetry-related molecules. The tetraplex molecules stack on one another in opposite polarity (head-to-head or tail-to-tail) to form a pseudocontinuous column. All of the 5'-end uridines rotate around the backbone of G2, swing out, and form unique octaplexes with the neighboring G tetraplexes, whereas the 3'-end uridines are stacked-in and form uridine tetrads. All of the bases are anti, and the riboses are in the mixed C2'- and C3'-puckering mode. Strontium ions are observed in every other guanine tetrad plane, sitting on the fourfold axis and associated to the eight 06 atoms of neighboring guanine bases in a bipyramidal-antiprism geometry. The hydrogens are clearly observed in the structure. ultra-high resolution | uridine tetrad | octad

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 [Angstrom] resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding.

The pp70/85-kDa S6 kinases, collectively referred to as [pp70.sup.S6k], are thought to participate in transit through the [G.sub.1] phase of the cell cycle. [pp70.sup.S6k] regulates the phosphorylation of the 40S ribosomal protein S6 and the transcription factor [CREM.sub.tau] [pp70.sup.S6k] is regulated by serine/threonine phosphorylation, and although 1-phosphatidylinositol 3-kinase and phospholipase C have been implicated as upstream regulators, the mechanism of activation and identity of the upstream [pp70.sup.S6k] kinases remain unknown. To improve our understanding of how this mitogen-stimulated protein kinase is regulated by growth factors and the immunosuppressant rapamycin, we have initiated a structure/function analysis of [pp70.sup.S6k] Our results indicate that both the N and C termini participate in the complex regulation of [pp70.sup.S6k] activity.

Data from several assay and immunochemical studies confirm that the NPI-1/SRP-1 human protein, whose function was unknown, is the alpha-subunit of the cytosolic protein complex karyopherin. Both the alpha- and beta-subunits of karyopherin function together in the docking of a nuclear localization sequence-containing substrate in digitonin-permeabilized cells' nuclear envelope. The absence of any one of the subunits inhibits the docking process.

Identification, synthesis, ligand-binding analysis, and NMR studies performed for two subdomains, peptide B and C, of the regulatory domain of protein kinase C(PKC) help determine the structure and phorbol ester-binding activity of these subdomains. These peptides have a high binding-affinity for phorbol ester with normal behavior in solution undergoing no concentration-dependent changes. Conformational ordering of peptide B takes places only in the presence of phospholipid. Hence PKC regulatory domain exhibits activity only on association with the lipid bilayer, which contains its activator.

A series of nonapeptide bombesin (BN) antagonists containing C-terminal Leu-psi(CH2-N)-Tac-NH2, Leu-psi(CH2-N)-MeTac-NH2 or Leu-psi(CH2-N)-Me2Tac-NH2 are synthesized upon incubation with formaldehyde or acetaldehyde. Binding affinities of the analogs varied in the order of (psi,CH2-N,Tac14)BN-(6-14) > (psi,CH2-N,Me2 Tac14)BN-(6-14) > (psi,CH2-N,MeTac14)BN-(6-14). Analogs with C-terminal Leu-psi(CH2-N)-Tac-NH2 are stronger growth inhibitors of 3T3 cells than those with C-terminal Leu-psi(CH2-N)-Me2TacNH2 or Leu-psi(CH2-N)-MeTac-NH2.

Site-directed mutagenetic studies of the transmembrane segment S6 of domain IV (IVS6) in the sodium ion-channel reveals that the intracellular end of this segment contributes to fast channel inactivation. This segment is present in the hydrophobic receptor site for the fast inactivation gate. The entry to and stability of the inactivated state are maintained by three hydrophobic amino acid residues at sites 1774-1776 in the IVS6 segment.

A new functional chimeric primase is constructed such that its 81 amino-end residues are derived from the phage T3 primase and the 484 carboxyl-end residues are derived from the phage T7 helicase. This protein has a one-third primase activity of T7 primase and uses identical recognition sites and oligoribonucleotides obtained from these sites. The chimeric and T7 proteins conserve the residues crucial for sequence 3'-CTG-5' interactions.

A purification technique that includes extraction with apolar solvent and adsorption chromatography helps purify a lipase from Euphorbia characias latex. On elution from silica gel complexed with a lipid, the 38-kDa, principal component becomes inactive when separated from the lipid fraction and regains 72% of its lipolytic activity when the protein and lipid components are added together. The apolipase exhibits amino acid homology and comparable lipase activity with the B chain of recin up to 23 residues. The lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly forms a part of the ricin B chain primary structure. Ricin B chain reacts with diethyl p-nitrophenyl phosphate, suggesting the presence of a serine residue and that it may be a galactosyl diacylglycerol lipase that resembles the E. characias lipase.

RNA helices that reconstruct the acceptor-TpsiC stem and the hybrid duplexes that reconstruct acceptor stem of methionine tRNA were studied for their aminoacylation with methionine. Observations of labeled RNA substrate on acidic polyacrylamide gel verified a low aminoacylation rate with RNA hairpin helices and duplexes that possessed sequences derived from the acceptor stem of tRNA(super met). The results indicated that specific aminoacylation can occur with methionine of small duplex substrates even without the anticodon or other RNA domains. A specific adaptation of methionine-tRNA synthetases to the acceptor end of tRNA(super met) was suggested based on the results.

This article discusses the application of structural genomics to proteins controling metal homeostasis in general and copper homeostasis proteins in particular. The research strategy focuses on CopC protein from Pseudomonas syringae and its structural analyses.

Structural genomics focusing on protein structure-function determination benefits from nuclear magnetic resonace methodology. It enables to analyze protein domains in releation to its structure and role in the overall function of the protein.

We have identified a nuclear-encoded Hb from plants (GLB3) that has a central domain similar to the 'truncated' Hbs of bacteria, protozoa, and algae. The three-dimensional structure of these Hbs is a 2-on-2 arrangement of [Alpha]-helices, distinct from the 3-on-3 arrangement of the standard globin fold [Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L. & Bolognesi, M. (2000) EMBOJ. 19, 2424-2434]. GLB3-like genes are not found in animals or yeast, but our analysis reveals that they are present in a wide range of Angiosperms and a Bryophyte. Although cyanobacteria and Chlamydomonas have 2-on-2 Hbs (GLBN), GLB3 is more likely related to GLBO-type 2-on-2 Hbs from bacteria. Consequently, GLB3 is unlikely to have arisen from a horizontal transfer between the chloroplast and nuclear genomes. Arabidopsis thaliana GLB3 protein exhibits unusual concentration-independent binding of [O.sub.2] and CO. The absorbance spectrum of deoxy-GLB3 is unique; the protein forms a transient six-coordinate structure after reduction and deoxygenation, which slowly converts to a five-coordinate structure. In A. thaliana, GLB3 is expressed throughout the plant but responds to none of the treatments that induce plant 3-on-3 Hbs. Our analysis of the sequence, ligand interactions, and expression profile of GLB3 indicates that this protein has unique biochemical properties, evolutionary history, and, most likely, a function distinct from those of other plant Hbs.

The successful prediction of protein structure from amino acid sequence requires two features: an efficient conformational search algorithm and an energy function with a global minimum in the native state. As a step toward addressing both issues, a threading-based method of secondary and tertiary restraint prediction has been developed and applied to ab initio folding. Such restraints are derived by extracting consensus contacts and local secondary structure from at least weakly scoring structures that, in some cases, can lack any global similarity to the sequence of interest. Furthermore, to generate representative protein structures, a reduced lattice-based protein model is used with replica exchange Monte Carlo to explore conformational space. We report results on the application of this methodology, termed TOUCHSTONE, to 65 proteins whose lengths range from 39 to 146 residues. For 47 (40) proteins, a cluster centroid whose rms deviation from native is below 6.5 (5) [Angstrom] is found in one of the five lowest energy centroids. The number of correctly predicted proteins increases to 50 when atomic detail is added and a knowledge-based atomic potential is combined with clustered and nonclustered structures for candidate selection. The combination of the ratio of the relative number of contacts to the protein length and the number of clusters generated by the folding algorithm is a reliable indicator of the likelihood of successful fold prediction, thereby opening the way for genome-scale ab initio folding.

Selectins mediate rolling of leukocytes by rapid formation and dissociation of selectin-ligand bonds, which are assumed to require high mechanical strength to prevent premature dissociation by the forces applied in shear flow. This assumption is based largely on the observation that increasing wall shear stress increases only modestly the dissociation of transient leukocyte tethers on very low selectin densities. P-selectin binds to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. Both PSGL-1 and P-selectin are extended homodimers. We perfused transfected cells expressing wild-type dimeric PSGL-1 or a chimeric monomeric form of PSGL-1 on immobilized dimeric or monomeric forms of P-selectin. Cells expressing dimeric or monomeric PSGL-1 tethered to P-selectin at equivalent rates. However, cells expressing dimeric PSGL-1 established more stable rolling adhesions, which were more shear resistant and exhibited less fluctuation in rolling velocities. On low densities of dimeric P-selectin, increasing wall shear stress more rapidly increased transient tether dissociation of cells expressing monomeric PSGL-1 than dimeric PSGL-1. Tether dissociation on low densities of monomeric P-selectin was even more shear sensitive. We conclude that dimerization of both PSGL-1 and P-selectin stabilizes tethering and rolling, probably by increasing rebinding within a bond cluster. Because transient tethers may have more than one bond, the mechanical strength of selectin-ligand bonds is likely to be lower than initially estimated. Tether strength may rely more on bond clusters to distribute applied force.

Sensory rhodopsins (SRs) belong to a subfamily of heptahelical transmembrane proteins containing a retinal chromophore. These photoreceptors mediate the cascade of vision in animal eyes and phototaxis in archaebacteria and unicellular flagellated algae. Signal transduction by these photoreceptors occurs by means of transducer proteins. The two archaebacterial sensory rhodopsins SRI and SRII are coupled to the membrane-bound Htrl and Htrll transducer proteins. Activation of these proteins initiates phosphorylation cascades that modulate the flagellar motors, resulting in either attractant (SRI) or repellent (SRII) phototaxis. In addition, transducer-free SRI and SRII were shown to operate as proton pumps, analogous to bacteriorhodopsin. Here, we present the x-ray structure of SRII from Natronobacterium pharaonis (pSRII) at 2.1-[Angstrom] resolution, revealing a unique molecular architecture of the retinal-binding pocket. In particular, the structure of pSRII exhibits a largely unbent conformation of the retinal (as compared with bacteriorhodopsin and halorhodopsin), a hydroxyl group of Thr-204 in the vicinity of the Schiff base, and an outward orientation of the guanidinium group of Arg-72. Furthermore, the structure reveals a putative chloride ion that is coupled to the Schiff base by means of a hydrogen-bond network and a unique, positively charged surface patch for a probable interaction with Htrll. The high-resolution structure of pSRII provides a structural basis to elucidate the mechanisms of phototransduction and color tuning.