The use of the Rx spin label in orientation measurement on proteins, by EPR.

The bipedal spin label Rx is more restricted in its conformation and dynamics than its monopodal counterpart R1. To systematically investigate the utility of the Rx label, we have attempted to comprehensively survey the attachment of Rx to protein secondary structures. We have examined the formation, structure and dynamics of the spin label in relation to the underlying protein in order to determine feasibility and optimum conditions for distance and orientation measurement by pulsed EPR. The labeled proteins have been studied using molecular dynamics, CW EPR, pulsed EPR distance measurement at X-band and orientation measurement at W-band. The utility of different modes and positions of attachment have been compared and contrasted.

The high-resolution crystal structure of a parallel-stranded guanine tetraplex.

Repeat tracts of guanine bases found in DNA and RNA can form tetraplex structures in the presence of a variety of monovalent cations. Evidence suggests that guanine tetraplexes assume important functions within chromosomal telomeres, immunoglobulin switch regions, and the human immunodeficiency virus genome. The structure of a parallel-stranded tetraplex formed by the hexanucleotide d(TG4T) and stabilized by sodium cations was determined by x-ray crystallography to 1.2 angstroms resolution. Sharply resolved sodium cations were found between and within planes of hydrogen-bonded guanine quartets, and an ordered groove hydration was observed. Distinct intra- and intermolecular stacking arrangements were adopted by the guanine quartets. Thymine bases were exclusively involved in making extensive lattice contacts.

Protein modules.

As the database of protein sequences grows it is becoming apparent that many proteins are constructed from relatively few modular units that appear many times. Determination of the three-dimensional structure of such modules by NMR has been possible due to their production in relatively large quantities by recombinant DNA techniques. The knowledge gained about the structure of individual modules can then be used to predict their properties in a variety of intact proteins.

Micro-CT for saw mark analysis on human bone.

In toolmark analysis, microscopy techniques, such as micro-CT, are used to visualise and measure toolmarks left on bones by a tool. In dismemberment cases, properties such as the width of the saw mark can provide cues to which tool was used by the culprit. The aim of the current study was to establish whether; (i) micro-CT is an appropriate imaging technique for saw mark analysis, (ii) toolmarks statistically differ when created with different tools, (iii) toolmark width can predict tool blade width, and (iv) toolmarks differ if created under different methodological conditions. Across two experiments, 270 saw marks were created using eight tools with either a controlled or free saw action on either fleshed or defleshed human long bone. Toolmarks were micro-CT scanned and seven toolmark properties were categorised or measured by two independent raters. The current study found that; (i) micro-CT was found to be a powerful and reliable imaging method for the visualisation and measurement of saw mark properties, (ii) toolmark properties differed significantly within and between various methodological conditions (p<.001) when created by eight different tools, (iii) a regression model developed using toolmark widths from Experiment 2 overall predicted 94% of tool widths in Experiment 1, and iv) methodological factors such as tissue presence and saw action significantly and inconsistently influenced toolmark properties for different tools. The study further validates the use of mirco-CT for saw mark analysis and demonstrates the potential of using toolmark properties to determine the tool used in cases of dismemberment. Given the effects that methodological factors such as tissue presence can have on toolmark properties, future studies should use experimental set ups with fleshed human tissue and use a free saw action.

The cutting edge - Micro-CT for quantitative toolmark analysis of sharp force trauma to bone.

Toolmark analysis involves examining marks created on an object to identify the likely tool responsible for creating those marks (e.g., a knife). Although a potentially powerful forensic tool, knife mark analysis is still in its infancy and the validation of imaging techniques as well as quantitative approaches is ongoing. This study builds on previous work by simulating real-world stabbings experimentally and statistically exploring quantitative toolmark properties, such as cut mark angle captured by micro-CT imaging, to predict the knife responsible. In Experiment 1 a mechanical stab rig and two knives were used to create 14 knife cut marks on dry pig ribs. The toolmarks were laser and micro-CT scanned to allow for quantitative measurements of numerous toolmark properties. The findings from Experiment 1 demonstrated that both knives produced statistically different cut mark widths, wall angle and shapes. Experiment 2 examined knife marks created on fleshed pig torsos with conditions designed to better simulate real-world stabbings. Eight knives were used to generate 64 incision cut marks that were also micro-CT scanned. Statistical exploration of these cut marks suggested that knife type, serrated or plain, can be predicted from cut mark width and wall angle. Preliminary results suggest that knives type can be predicted from cut mark width, and that knife edge thickness correlates with cut mark width. An additional 16 cut marks walls were imaged for striation marks using scanning electron microscopy with results suggesting that this approach might not be useful for knife mark analysis. Results also indicated that observer judgements of cut mark shape were more consistent when rated from micro-CT images than light microscopy images. The potential to combine micro-CT data, medical grade CT data and photographs to develop highly realistic virtual models for visualisation and 3D printing is also demonstrated. This is the first study to statistically explore simulated real-world knife marks imaged by micro-CT to demonstrate the potential of quantitative approaches in knife mark analysis. Findings and methods presented in this study are relevant to both forensic toolmark researchers as well as practitioners. Limitations of the experimental methodologies and imaging techniques are discussed, and further work is recommended.

Molecular chaperones: small heat shock proteins in the limelight.

Small heat shock proteins have been the Cinderellas of the molecular chaperone world, but now the crystal structure of a small heat shock protein has been solved and mutation of two human homologues implicated in genetic disease. Intermediate filaments appear to be one of the key targets of their chaperone activity.

3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase.

BACKGROUND: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2). RESULTS: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. CONCLUSIONS: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

hSRY: molecular gender bender.

The recently published solution structure of the DNA-binding domain of hSRY in complex with a DNA octamer offers insight into the sex reversal effects of mutations in hSRY and the interaction of all HMG boxes with DNA.

Characterisation of the interaction between PCNA and Gadd45.

We have examined the interaction between the DNA replication and repair protein PCNA, and the growth arrest and DNA damage induced protein Gadd45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA but in reciprocal experiments, an anti-C terminal anti-PCNA antibody failed to co-immunoprecipitate Gadd45. We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pombe PCNA. We have determined that the N-terminal 94 amino acids of Gadd45 bind to PCNA, and using a series of N-terminal and C-terminal deletions of human PCNA we have mapped two potential Gadd45 binding sites. Deletion of the last 6 amino acids of PCNA ablated interaction, suggesting a role in Gadd45 binding. This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45. Using a peptide ELISA approach, we showed that Gadd45 protein binds strongly to three regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to residues 121-170. The crystal structure of PCNA provides insight into our genetic and immunochemical data. Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support for the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.

Binding of U1A protein to the 3' untranslated region of its pre-mRNA.

We have studied the global structure of the U1A 3' untranslated region (UTR) element using fluorescence resonance energy transfer. Comparison of a single UTR-box with a series of oligoadenine bulges indicates that the UTR-box introduces a significant kink into the axis of the RNA, and quantification of the results suggests an included bend angle of approximately 100 degrees (i.e. 80 degrees from linear). The complete 3'-UTR element is also severely kinked by the two UTR-boxes. We can observe binding of U1A protein to the 3'-UTR element by a change in the fluorescence anisotropy of Cy3 attached to one of the helical ends. In parallel with the binding, we observe a marked increase in fluoresence resonance energy transfer efficiency between fluorophores attached at the two 5' termini, indicating a significant change in global conformation induced by the binding of the protein.

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

Solution structure of a parallel-stranded tetraplex formed by d(TG4T) in the presence of sodium ions by nuclear magnetic resonance spectroscopy.

DNA sequences containing repetitive oligo-guanine sequences are observed in telomeric DNA and in recombination hotspots. In vitro, these sequences fold into at least two classes of unusual conformations involving the formation of guanine tetrads (G-quartets). Through the use of NMR, we have established that in solution in the presence of sodium ions, the oligodeoxynucleotide dTG4T forms a parallel-stranded tetraplex, with hydrogen-bonded guanine tetrads. Here we present the detailed evidence for this configuration in 100 mM sodium ions at neutral pH. We show that the parallel-stranded tetraplex structure is stabilised relative to the single-strands when Na ions are replaced by K ions, without any evidence of significant change in those chemical shifts associated with the tetraplex form. We also present a model for the Na+ solution structure of the tetraplex, by back-calculation from NOE volumes. We obtain excellent agreement with the data for structures with characteristics similar to B-DNA for the internal tetrads, G3 and G4. The outer tetrads, particularly the 5'-terminal G2, display some unstacking. We see no evidence for formation of thymine tetrads analogous to the uridine tetrads observed in RNA, but rather we find the terminal thymine conformations are not well determined by our data. We observe an uninterrupted cylindrical channel through the centre of the complex.

Severe axial bending of RNA induced by the U1A binding element present in the 3' untranslated region of the U1A mRNA.

The 3' untranslated region of the U1A mRNA contains a binding site for the U1A protein that consists of two asymmetric internal bulges. The bulges each comprise a loop of seven unpaired bases opposing a single base (termed a U1A box). The seven-base loops are located on opposite strands, distributed in a symmetrical manner about the intervening four-base duplex. We have investigated the global conformation of this binding element. Comparison of electrophoretic mobilities of RNA duplexes interrupted by a single U1A box with a series of duplexes of the same length containing oligoadenine bulges indicates that the individual boxes cause a substantial kinking of the helix axis, estimated to be 90 (+/- 10) degrees. A series of RNA duplexes were constructed containing a U1A box separated from an A5 bulge by a duplex section of length between 3 and 21 bp. It was found that the electrophoretic mobilities of these species varied sinusoidally, indicating that the U1A box introduces a defined kink into the RNA helix, rather than a point of flexibility. Electrophoretic experiments with the complete U1A binding element suggest that the axial trajectories of the two U1A boxes combine to give an approximately in-line, 180 degrees change in duplex direction.

The A730 loop is an important component of the active site of the VS ribozyme.

The core of the VS ribozyme comprises five helices, that act either in cis or in trans on a stem-loop substrate to catalyse site-specific cleavage. The structure of the 2-3-6 helical junction indicates that a cleft is formed between helices II and VI that is likely to serve as a receptor for the substrate. Detailed analysis of sequence variants suggests that the base bulges of helices II and VI play an architectural role. By contrast, the identity of the nucleotides in the A730 loop is very important for ribozyme activity. The base of A756 is particularly vital, and substitution by any other nucleotide or ablation of the base leads to a major reduction in cleavage rate. However, variants of A756 bind substrate efficiently, and are not defective in global folding. These results suggest that the A730 loop is an important component of the active site of the ribozyme, and that A756 could play a key role in catalysis.

Solution structure of a pair of complement modules by nuclear magnetic resonance.

A portion of human complement factor H spanning the 15th (H15) and 16th (H16) of its 20 modules, has been expressed in a yeast vector and subjected to structure determination in solution using two-dimensional 1H-NMR. The structure of H15 is very similar to that already established for the fifth module of factor H and H16, consistent with the view that all such complement control (C-) modules share a common overall topology. In addition, the tertiary structures of the component modules of the H15-16 pair are very similar to those of the modules when expressed individually, implying that each folds entirely autonomously within intact factor H. Aromatic residues in the third turn of H15 and the second turn of H16, together with a leucine residue from the linker region, contribute to a small intermodular interface. Comparatively few nuclear Overhauser effects were observable between protons on different modules. Consequently, a wide range of angles of "twist" (131 (+/- 146) degrees, mean value (+/- 1 standard deviation)), i.e. rotation about the long axis of one module with respect to the other, exists in the family of structures generated on the basis of the experimental data. However, much smaller variations occur in the two, orthogonal, angles (175 (+/- 12) degrees and 103 (+/- 6) degrees) that describe the "tilt". These observations may suggest upper limits on the relative flexibility of the two modules. Models were built to assess the outcome of applying such restrictions to all the neighbours within a string of 20 C-modules, and the resulting structures compare well with factor H as visualized by electron microscopy.

Mutual correction of faulty PCNA subunits in temperature-sensitive lethal mus209 mutants of Drosophila melanogaster.

Proliferating cell nuclear antigen (PCNA) functions in DNA replication as a processivity factor for polymerases delta and epsilon, and in multiple DNA repair processes. We describe two temperature-sensitive lethal alleles (mus209(B1) and mus209(2735)) of the Drosophila PCNA gene that, at temperatures permissive for growth, result in hypersensitivity to DNA-damaging agents, suppression of position-effect variegation, and female sterility in which ovaries are underdeveloped and do not produce eggs. We show by mosaic analysis that the sterility of mus209(B1) is partly due to a failure of germ-line cells to proliferate. Strikingly, mus209(B1) and mus209(2735) interact to restore partial fertility to heteroallelic females, revealing additional roles for PCNA in ovarian development, meiotic recombination, and embryogenesis. We further show that, although mus209(B1) and mus209(2735) homozygotes are each defective in repair of transposase-induced DNA double-strand breaks in somatic cells, this defect is substantially reversed in the heteroallelic mutant genotype. These novel mutations map to adjacent sites on the three-dimensional structure of PCNA, which was unexpected in the context of this observed interallelic complementation. These mutations, as well as four others we describe, reveal new relationships between the structure and function of PCNA.

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.

The orientation of a tandem POTRA domain pair, of the beta-barrel assembly protein BamA, determined by PELDOR spectroscopy.

The outer membrane beta-barrel trans-membrane proteins in gram-negative bacteria are folded into the membrane with the aid of polypeptide transport-associated (POTRA) domains. These domains occur, and probably function, as a tandem array situated on the periplasmic side of the outer membrane. Two crystal structures and one NMR study have attempted to define the structure and articulation of the POTRA domains of the Escherichia coli, prototypic Omp85 protein BamA. We have used pulsed electron paramagnetic resonance (EPR) to determine the distance and distance distribution between (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSSL), placed across the domain interface of the first two POTRA domains of BamA. Our results show tightly defined interdomain distance distributions that indicate a well-defined domain orientation. Examination of the known structures revealed that none of them fitted the EPR data. A combination of EPR and NMR data was used to generate converged structures with defined domain-domain orientation.

The Holliday junction is finally seen with crystal clarity.

After a long wait, we finally see the structure of the Holliday junction of genetic recombination at atomic resolution. It comprises a right-handed cross of DNA molecules, with an antiparallel orientation of strands.