Hairpins play a central role in numerous protein folding and misfolding scenarios. Prior studies of hairpin folding, many conducted with analogs of a sequence from the B1 domain of protein G, suggest that faster folding can be achieved only by optimizing the turn propensity of the reversing loop. Based on studies using dynamic NMR, the native GB1 sequence is a slow folding hairpin (k F 278 ‫؍‬ 1.5 ؋ 10 4 ͞s). GB1 hairpin analogs spanning a wide range of thermodynamic stabilities (⌬G U 298 ‫؍‬ ؊3.09 to ؉3.25 kJ͞mol) were examined. Fold-stabilizing changes in the reversing loop can act either by accelerating folding or retarding unfolding; we present examples of both types. The introduction of an attractive sidechain͞side-chain Coulombic interaction at the chain termini further stabilizes this hairpin. The 1.9-fold increase in folding rate constant observed for this change at the chain termini implies that this Coulombic interaction contributes before or at the transition state. This observation is difficult to rationalize by ''zipper'' folding pathways that require native turn formation as the sole nucleating event; it also suggests that Coulombic interactions should be considered in the design of systems intended to probe the protein folding speed limit.␤-hairpin ͉ exchange broadening ͉ folding dynamics ͉ loop search P rotein engineering experiments indicate that ␤-hairpins appear as transition-state features in numerous folding pathways. Hairpin redesign has resulted in changes in protein-folding mechanisms (1), and hairpin stabilization can accelerate (1-3) or retard (2, 4) protein folding. The protein-folding problem continues to be of high interest for at least three reasons: predicting structures from genome-derived sequences, improving a priori protein-fold design, and enhancing our understanding of the mechanisms of protein misfolding diseases (6, 7). Folding rates have been of particular interest, with more examples of redesigned proteins that fold near the calculated protein-folding speed limit (8) appearing regularly. For ␤-sheet proteins and ␤ oligomers (as found in amyloid fibrils formed from misfolded protein states), hairpin dynamics play a key role.Although there is an increasing body of data on hairpin folding dynamics (9-15), with one exception these have not included a set of probing mutations to address specific questions. That exception (14) suggests that loops with a greater turn preference accelerate folding to a much greater extent than the optimization of hydrophobic interactions in the folded state. Other data (12) suggest that the length of the loop connecting the hydrophobic residues that form hairpin-stabilizing cross-strand interactions is reflected in the folding rate. Throughout, hairpin folding has been modeled as a two-state equilibrium. Whether hairpin͞coil transitions are 1-s versus 50-s events has significant consequences. Peptide helix nucleation is a sub-s event (10,16,17), which allows helix formation to be a preequilibrium event relative to the hydrophobic-collapse stage o...

Tetraploid cotton is one of the most extensively cultivated species. Two tetraploid species, Gossypium hirsutum L. and G. barbadense L., dominate the world's cotton production. To better understand the genetic basis of cotton fibre traits for the improvement of fibre quality, a genetic linkage map of tetraploid cotton was constructed using sequence-related amplified polymorphisms (SRAPs), simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs). A total of 238 SRAP primer combinations, 368 SSR primer pairs and 600 RAPD primers were used to screen polymorphisms between G. hirsutum cv. Handan208 and G. barbadense cv. Pima90 which revealed 749 polymorphic loci in total (205 SSRs, 107 RAPDs and 437 SRAPs). Sixty-nine F 2 progeny from the interspecific cross of ÔHandan208Õ · ÔPima90Õ were genotyped with the 749 polymorphic markers. A total of 566 loci were assembled into 41 linkage groups with at least three loci in each group. Twenty-eight linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 5141.8 cM with a mean interlocus space of 9.08 cM. A v 2 test for significance of deviations from the expected ratio (1 : 2 : 1 or 3 : 1) identified 135 loci (18.0%) with skewed segregation, most of which had an excess of maternal parental alleles. In total, 13 QTL associated with fibre traits were detected, among which two QTL were for fibre strength, four for fibre length and seven for micronaire value. These QTL were on nine linkage groups explaining 16.18-28.92% of the trait variation. Six QTL were located in the A subgenome, six QTL in the D subgenome and one QTL in an unassigned linkage group. There were three QTL for micronaire value clustered on LG1, which would be very useful for improving this trait by molecular marker-assisted selection.

The crystal structure of chalcopyrite, CuFeS2, has been refined to an R value of 0.031 using multiple sets of 4-circle diffractometer data and full-matrix least-squares procedures. Spherical absorption corrections and anomalous dispersion data were applied in the refinement of the sulphur x coordinate at site 8d [0.2574 (2), ¼, -~] and the anisotropic temperature factors. The thermal motion of all atoms is essentially isotropic and in excellent agreement with that of equivalent atoms in cubanite, CuFezSa. The metal-sulphur distances of Cu-S=2-302 (1) and Fe-S=2.257 (1) A, are significantly closer than those reported previously. There is stereochemical evidence that the structure exists in a strong covalently-bonded configuration which has an effective ionic state between Cu + Fe 3 + $22-and Cu 2 + Fe z+ $2 2-.

Summary p53 protein was detected by immunohistochemistry in 42% of 52 colorectal adenocarcinomas. Positive tumours were significantly more frequent in the distal colon, and demonstrated a higher rate of cell proliferation. No correlation was found with tumour grade, Dukes' stage, presence of DNA aneuploidy or patient survival. The role of p53 in colorectal carcinogenesis is discussed with particular reference to differences between proximal and distal large bowel cancers. p53 is a 53kD nuclear protein, highly conserved in vertebrates, which is believed to regulate entry into and progression through the normal cell cycle (Mercer et al., 1984). Like c-myc it is induced during transition from Go to G, phase (Milner & Milner, 1981;Reich & Levine, 1984), and is present at low levels in most normal fetal and adult tissues (Rogel et al., 1985). Studies of p53 expression in cultured cells suggest that increased levels are associated either with an abnormal mutated protein (Finlay et al., 1988), or stablisation of the protein in a complex with viral antigens, e.g. SV40 large T antigen (Lane & Crawford, 1979). Point mutations occurring in a highly conserved region of the gene are known to activate p53 in the primary rat embryo fibroblast transfection assay for dominant oncogenes , whereas the wild type protein has a tumour suppressor action . The presence of increased levels of protein may therefore provide a marker for mutated p53.Elevated p53 expression has been described in a number of human tumours including carcinoma of the breast (Crawford et al., 1984;Cattoretti et al., 1988;Thompson et al., 1990), colorectum (Crawford et al., 1984), and lung (Iggo et al., 1990). Colorectal cancer is characterised by frequent deletion of chromosome 17p close to the p53 locus (Vogelstein et al., 1988), and elevated protein levels have been found by radioimmunoassay in 44% of tumours (Crawford et al., 1984). These studies suggest that in some tumours hemizygous deletion of one p53 allele is accompanied by mutation and overexpression of the other. Recently van den Berg et al. (1989) described the immunohistochemical detection of p53 in 55% of 29 colorectal cancers. However no information was given regarding the relationship of p53 expression to clinico-pathological variables several of which are believed to correlate with the biological aggressiveness and stage of progression of a tumour. The aim of the present study was to assess these relationships in a larger series and investigate the value of the immunocytochemical detection of p53 as a prognostic indicator. Materials and methodsFresh tumour tissue was obtained from 52 adenocarcinomas of the large bowel from 52 patients. A single 4 p frozen section was cut from each tumour, air dried overnight, and fixed for 15 min in acetone at 4'C.In five cases tissue was available from up to five different areas of the same tumour. These were included in the main series in order to assess the effect of intra-tumour heterogeneity on the detection of p53.Sections were incubated with Pab421, ...

Gai and co-workers (Bunagan, M. R. (2006) J. Phys. Chem. B 110, 3759-3763) reported computational design studies suggesting that a D9E mutation would stabilize the Trp-cage. Experimental studies for this mutation were reported in 2008 (Hudaky, P. (2008) Biochemistry 47, 1007-1016); the authors suggested that [D9E]-TC5b presented a more compact, and melting resistant structure due to the “optimal distance between the two sides of the molecule”. Nonetheless, the authors reported essentially the same CD melting temperature, 38±0.3 °C, for TC5b and its [D9E] mutant. In this study, a more stable Trp-cage, DAYAQ WLKDG GPSSG RPPPS, was examined by NMR and CD with the following mutations: [D9E], [D9R,R16E], [R16Orn], [D9E,R16Orn], [R16K], and [D9E,R16K]. Of these, the [D9E]-mutant displayed the smallest acidification induced change in the apparent Tm. In analogy to the prior study, the CD melts of TC10b and its [D9E] mutant were, however, very similar; all of the other mutations were significantly fold destabilizing by all measures. A detailed analysis indicates that the original D9/R16 salt bridge is optimal with regard to fold cooperativity and fold stabilization. Evidence for salt-bridging is also provided for a swapped pair, the [D9R,R16E]-mutant. Model systems reveal that an ionized aspartate at the C-terminus of a helix significantly decreases intrinsic helicity, a requirement for Trp-cage fold stability. The CD evidence which was cited as supporting increased fold stability for the [D9E]-TC5b at higher temperatures appears to be a reflection of increased helix stability in both the folded and unfolded state rather than a more favorable salt bridge. The present study also provides evidence for other Trp-cage stabilizing roles of the R16 sidechain.