The flexibility in the proline ring couples to the protein backbone

In proteins, the proline ring exists predominantly in two discrete states. However, there is also a small but significant amount of flexibility in the proline ring of high-resolution protein structures. We have found that this side-chain flexibility is coupled to the backbone conformation. To study this coupling, we have developed a model that is simply based on geometric and steric factors and not on energetics. We show that the coupling between phi and chi1 torsions in the proline ring can be described by an analytic equation that was developed by Bricard in 1897, and we describe a computer algorithm that implements the equation. The model predicts the observed coupling very well. The strain in the C(gamma)-C(delta)-N angle appears to be the principal barrier between the UP and DOWN pucker. This strain is relaxed to allow the proline ring to flatten in the rare PLANAR conformation.     

Center of pressure and center of mass behavior during gait initiation on inclined surfaces: A statistical parametric mapping analysis

This study analyzed gait initiation (GI) on inclined surfaces with 68 young adult subjects of both sexes. Ground reaction forces and moments were collected using two AMTI force platforms, of which one was in a horizontal position and the other was inclined by 8% in relation to the horizontal plane. Departing from a standing position, each participant executed three trials in the following conditions: horizontal position (HOR), inclined position at ankle dorsi-flexion (UP), and inclined position at ankle plantar-flexion (DOWN). Statistical parametric mapping analysis was performed over the entire center of pressure (COP) and center of mass (COM) time series. COP excursion did not show significant differences in the medial-lateral (ML) direction in both inclined conditions, but it was greater in the anterior-posterior (AP) direction for both inclined conditions. COP velocities are smaller in discrete portions of GI for the UP and DOWN conditions. COM displacement was greater in the ML direction during anticipatory postural adjustments (APA) in the UP condition, and COM moves faster in the ML direction during APA in the UP condition but slower at the end of GI for both the UP and the DOWN conditions. The COP-COM vector showed a greater angle in the DOWN condition. We observed changes for COP and COM in GI in both the UP and the DOWN conditions, with the latter showing changes for a great extent of the task. Both the UP and the DOWN conditions showed increased COM displacement and velocity. The predominant characteristic during GI on inclined surfaces, including APA, appears to be the displacement of the COM.     

UP States Protect Ongoing Cortical Activity from Thalamic Inputs

Cortical neurons in vitro and in vivo fluctuate spontaneously between two stable membrane potentials: a depolarized UP state and a hyperpolarized DOWN state. UP states temporally correspond with multineuronal firing sequences which may be important for information processing. To examine how thalamic inputs interact with ongoing cortical UP state activity, we used calcium imaging and targeted whole-cell recordings of activated neurons in thalamocortical slices of mouse somatosensory cortex. Whereas thalamic stimulation during DOWN states generated multineuronal, synchronized UP states, identical stimulation during UP states had no effect on the subthreshold membrane dynamics of the vast majority of cells or on ongoing multineuronal temporal patterns. Both thalamocortical and corticocortical PSPs were significantly reduced and neuronal input resistance was significantly decreased during cortical UP states – mechanistically consistent with UP state insensitivity. Our results demonstrate that cortical dynamics during UP states are insensitive to thalamic inputs.     

Methods for predicting cortical UP and DOWN states from the phase of deep layer local field potentials

During anesthesia, slow-wave sleep and quiet wakefulness, neuronal membrane potentials collectively switch between de- and hyperpolarized levels, the cortical UP and DOWN states. Previous studies have shown that these cortical UP/DOWN states affect the excitability of individual neurons in response to sensory stimuli, indicating that a significant amount of the trial-to-trial variability in neuronal responses can be attributed to ongoing fluctuations in network activity. However, as intracellular recordings are frequently not available, it is important to be able to estimate their occurrence purely from extracellular data. Here, we combine in vivo whole cell recordings from single neurons with multi-site extracellular microelectrode recordings, to quantify the performance of various approaches to predicting UP/DOWN states from the deep-layer local field potential (LFP). We find that UP/DOWN states in deep cortical layers of rat primary auditory cortex (A1) are predictable from the phase of LFP at low frequencies (< 4 Hz), and that the likelihood of a given state varies sinusoidally with the phase of LFP at these frequencies. We introduce a novel method of detecting cortical state by combining information concerning the phase of the LFP and ongoing multi-unit activity.     

High pressure liquid chromatography purification of UP1 and UP2, two related single-stranded nucleic acid-binding proteins from calf thymus

Two single-stranded nucleic acid-binding proteins, UP1 and UP2, that were originally reported by Herrick and Alberts (Herrick, G., and Alberts, B. (1976) J. Biol. Chem. 251, 2124-2132) have been purified to apparent homogeneity from calf thymus by high performance liquid chromatography. The amino acid sequence of UP1 (Williams, K. R., Stone, K. L., LoPresti, M. B., Merrill, B. M., and Planck, S. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5666-5670) reveals that UP1 contains 195 amino acids, including one dimethylarginine residue near its COOH terminus. Further analysis of this sequence now demonstrates that UP1 contains a 91-residue internal repeat such that when residues 3-93 (the "A" region) are aligned with residues 94-194 (the "B" region), 32% of the amino acids in these two regions are identical and an additional 39% of those changes that are seen could be accomplished by single base changes. The high degree of internal homology between residues 51-61 and 143-152 and in particular the high density of aromatic and positively charged amino acids in these two regions suggest that residues 51-61 and 143-152 may constitute two independent DNA-binding sites. Solid-phase sequencing of three tryptic peptides that together account for 9% of the 39,500-dalton UP2 protein demonstrate that there is a high degree of sequence homology between UP1 and UP2. Of the 34 residues that have been sequenced in UP2, 44% are identical in both UP1 and UP2. The blocked NH2 terminus, amino acid composition, particularly with regard to its high glycine content and the presence of dimethylarginine, and molecular weight of UP2 suggest this protein is related to proteins that have previously been found associated with heterogeneous RNA. Taken together, these data indicate that both UP1 and UP2 belong to a new family of single-stranded nucleic acid-binding proteins that may be closely related to heterogeneous ribonucleoproteins.     

Gait stability,variability and complexity on inclined surfaces

This study evaluated the gait stability, variability, and complexity of healthy young adults on inclined surfaces. A total of 49 individuals walked on a treadmill at their preferred speed for 4 min at inclinations of 6%, 8%, and 10% in upward (UP) and downward (DOWN) conditions, and in horizontal (0%) condition. Gait variability was assessed using average standard deviation trunk acceleration between strides (VAR), gait stability was assessed using margin of stability (MoS) and maximum Lyapunov exponent (λs), and gait complexity was assessed using sample entropy (SEn). Trunk variability (VAR) increased in the medial-lateral (ML), anterior-posterior, and vertical directions for all inclined conditions. The SEn values indicated that movement complexity decreased almost linearly from DOWN to UP conditions, reflecting changes in gait pattern with longer and slower steps as inclination increased. The DOWN conditions were associated with the highest variability and lowest stability in the MoS ML, but not in λs. Stability was lower in UP conditions, which exhibited the largest λs values. The overall results support the hypothesis that inclined surfaces decrease gait stability and alter gait variability, particularly in UP conditions.     

Sleepy dialogues between cortex and hippocampus: who talks to whom?

During NREM sleep, neocortical neurons undergo near-synchronous transitions, every second or so, between UP states, during which they are depolarized and fire actively, and DOWN states, during which they are hyperpolarized and completely silent. In this issue of Neuron, Isomura et al. report that slow oscillations of membrane potential occur near-synchronously not only in neocortex but also in entorhinal cortex and subiculum. Within the hippocampus proper, pyramidal neurons lack the bistability of UP and DOWN states, but their firing is strongly modulated by cortical activity during the UP state. Intriguingly, many hippocampal neurons fire during the cortical DOWN state. Thus, during sleep UP states, the cortex can talk to the hippocampus, but it is unclear whether the hippocampus talks back.     

How Body Orientation Affects Concepts of Space,Time and Valence: Functional Relevance of Integrating Sensorimotor Experiences during Word Processing

The aim of the present study was to test the functional relevance of the spatial concepts UP or DOWN for words that use these concepts either literally (space) or metaphorically (time, valence). A functional relevance would imply a symmetrical relationship between the spatial concepts and words related to these concepts, showing that processing words activate the related spatial concepts on one hand, but also that an activation of the concepts will ease the retrieval of a related word on the other. For the latter, the rotation angle of participant’s body position was manipulated either to an upright or a head-down tilted body position to activate the related spatial concept. Afterwards participants produced in a within-subject design previously memorized words of the concepts space, time and valence according to the pace of a metronome. All words were related either to the spatial concept UP or DOWN. The results including Bayesian analyses show (1) a significant interaction between body position and words using the concepts UP and DOWN literally, (2) a marginal significant interaction between body position and temporal words and (3) no effect between body position and valence words. However, post-hoc analyses suggest no difference between experiments. Thus, the authors concluded that integrating sensorimotor experiences is indeed of functional relevance for all three concepts of space, time and valence. However, the strength of this functional relevance depends on how close words are linked to mental concepts representing vertical space.     

Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.     

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.     

Integration and segregation of activity in entorhinal-hippocampal subregions by neocortical slow oscillations

Brain systems communicate by means of neuronal oscillations at multiple temporal and spatial scales. In anesthetized rats, we find that neocortical "slow" oscillation engages neurons in prefrontal, somatosensory, entorhinal, and subicular cortices into synchronous transitions between UP and DOWN states, with a corresponding bimodal distribution of their membrane potential. The membrane potential of hippocampal granule cells and CA3 and CA1 pyramidal cells lacked bimodality, yet it was influenced by the slow oscillation in a region-specific manner. Furthermore, in both anesthetized and naturally sleeping rats, the cortical UP states resulted in increased activity of dentate and most CA1 neurons, as well as the highest probability of ripple events. Yet, the CA3-CA1 network could self-organize into gamma bursts and occasional ripples during the DOWN state. Thus, neo/paleocortical and hippocampal networks periodically reset, self-organize, and temporally coordinate their cell assemblies via the slow oscillation.     

The structure of 3'-O-anthraniloyladenosine, an analogue of the 3'-end of aminoacyl-tRNA.

3'-O-Anthraniloyladenosine, an analogue of the 3'- terminal aminoacyladenosine residue in aminoacyl-tRNAs, was prepared by chemical synthesis, and its crystal structure was determined. The sugar pucker of 3'-O-anthraniloyladenosine is 2'-endo resulting in a 3'-axial position of the anthraniloyl residue. The nucleoside is insynconformation, which is stabilized by alternating stacking of adenine and benzoyl residues of the neighboring molecules in the crystal lattice. The conformation of the 5'-hydroxymethylene in 3'-O- anthraniloyladenosine is gauche-gauche. There are two intramolecular and two intermolecular hydrogen bonds and several H-bridges with surrounding water molecules. The predominant structure of 3'-O-anthraniloyladenosine in solution, as determined by NMR spectroscopy, is 2'-endo,gauche-gauche and anti for the sugar ring pucker, the torsion angle around the C4'-C5'bond and the torsion angle around the C1'-N9 bond, respectively. The 2'-endo conformation of the ribose in 2'(3')-O-aminoacyladenosine, which places the adenine and aminoacyl residues in equatorial and axial positions, respectively, could serve as a structural element that is recognized by enzymes that interact with aminoacyl-tRNA or by ribosomes to differentiate between aminoacylated and non-aminoacylated tRNA.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

STDP in a bistable synapse model based on CaMKII and associated signaling pathways

The calcium/calmodulin-dependent protein kinase II (CaMKII) plays a key role in the induction of long-term postsynaptic modifications following calcium entry. Experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states. The biochemical network involving CaMKII and its regulating protein signaling cascade has been hypothesized to durably maintain the evoked synaptic state in the form of a bistable switch. However, it is still unclear whether experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such a network. We present a detailed biochemical model of the CaMKII autophosphorylation and the protein signaling cascade governing the CaMKII dephosphorylation. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentration, and high calcium transients can switch the system from the weakly phosphorylated (DOWN) to the highly phosphorylated (UP) state of the CaMKII (similar to a LTP event). We show here that increased CaMKII dephosphorylation activity at intermediate Ca²⁺ concentrations can lead to switching from the UP to the DOWN state (similar to a LTD event). This can be achieved if protein phosphatase activity promoting CaMKII dephosphorylation activates at lower Ca²⁺ levels than kinase activity. Finally, it is shown that the CaMKII system can qualitatively reproduce results of plasticity outcomes in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. This shows that the CaMKII protein network can account for both induction, through LTP/LTD-like transitions, and storage, due to its bistability, of synaptic changes.     

Structure of spontaneous UP and DOWN transitions self-organizing in a cortical network model

Synaptic plasticity is considered to play a crucial role in the experience-dependent self-organization of local cortical networks. In the absence of sensory stimuli, cerebral cortex exhibits spontaneous membrane potential transitions between an UP and a DOWN state. To reveal how cortical networks develop spontaneous activity, or conversely, how spontaneous activity structures cortical networks, we analyze the self-organization of a recurrent network model of excitatory and inhibitory neurons, which is realistic enough to replicate UP–DOWN states, with spike-timing-dependent plasticity (STDP). The individual neurons in the self-organized network exhibit a variety of temporal patterns in the two-state transitions. In addition, the model develops a feed-forward network-like structure that produces a diverse repertoire of precise sequences of the UP state. Our model shows that the self-organized activity well resembles the spontaneous activity of cortical networks if STDP is accompanied by the pruning of weak synapses. These results suggest that the two-state membrane potential transitions play an active role in structuring local cortical circuits.     

The role and predicted propensity of conserved proline residues in the 5-HT3 receptor

5-HT3 receptors possess a number of highly conserved proline residues. We changed each of these to alanine, expressed the mutants as homomeric 5-HT3A receptors in HEK293 cells, and analyzed them with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Pro56, Pro104, Pro123, and Pro170 resulted in ablation of radioligand binding, whereas mutation of Pro257 and Pro301 did not. Only the latter were expressed at the plasma membrane but were non-functional. Thus the former, which are in the N-terminal domain, may be involved in forming correct receptor structure, while those in the transmembrane region (Pro257 and Pro301) are necessary for the function of the protein. To explore the conformational preference (propensity) of these residues we examined the proportion of cis-prolines and the influence of adjacent residues in known protein structures. 4.7% of prolines in the protein data base were in the cis conformation, and the distribution of amino acids adjacent to cis-prolines was not randomly distributed. Comparison of the proportion of each amino acid residue adjacent to a cis-proline revealed that aromatic and bend-facilitating residues were favored while those with beta-branched chains were not. Thus five residues (Gly, Pro, Tyr, Trp, Phe) and three residues (Pro, Tyr, Phe) were found more frequently than expected before and after cis-prolines respectively, whereas five residues (Val, Ile, Leu, Asp, Thr) and two residues (Asp, Glu) were found less frequently. Of the 20 proline residues in the 5-HT3A receptor subunit only Pro170 has adjacent residues that are favorable. Mutating these to non-favorable residues resulted in ablation of ligand binding, whereas replacement with alternative favorable residues did not. We therefore propose that Pro170, which is part of the characteristic cys-loop found in this family of proteins, may be in the cis conformation.     

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.     

Cis peptide bonds in proteins: residues involved, their conformations, interactions and locations.

An analysis of a non-redundant set of protein structures from the Brookhaven Protein Data Bank has been carried out to find out the residue preference, local conformation, hydrogen bonding and other stabilizing interactions involving cis peptide bonds. This has led to a reclassification of turns mediated by cis peptides, and their average geometrical parameters have been evaluated. The interdependence of the side and main-chain torsion angles of proline rings provided an explanation why such rings in cis peptides are found to have the DOWN puckering. A comparison of cis peptides containing proline and non-proline residues show differences in conformation, location in the secondary structure and in relation to the centre of the molecule, and relative accessibilities of residues. Relevance of the results in mutation studies and the cis-trans isomerization during protein folding is discussed.     

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR structures of a nonapeptide from DNA binding domain of human polymerase-alpha determined by iterative complete-relaxation-matrix approach.

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from the DNA binding domain of human polymerase-alpha, were determined by complete relaxation matrix analysis of transverse NOE data. The structures exhibit a type III turn with residues KCPC, and the remaining residues exhibit non-ordered structures. The turn was confirmed by alpha, N (i,i+3) connectivity, a low temperature coefficient of NH chemical shift (-3.1 x 10(-3)) of the fourth residue, 3J(NHalpha) coupling constants, and characteristic CD peaks at 228 and 200 nm. Furthermore, phi and psi dihedral angles for the i + 1, and i + 2 residues of the turn are found to be -80 and -41 and -60 and -40 degrees. The first proline residue is trans- while the second exists in both cis- and trans- configurations, with trans- being more than 80% populated. The trans-configuration was established from C5alpha-P6alpha correlation and phi and psi angles of the proline. The five-membered proline ring is in DOWN puckered (C-beta-exo/C-gamma-endo) conformation. The structure of the peptide reveals that the two cysteine thiols are approximately 5 A(o) apart and appropriately positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used anti-cancer drug.