The influence of amino-acid sequence on protein structure

On the basis of the known sequences and structures of myoglobin, and alpha and beta hemoglobin, a possible correlation between certain amino acids in the sequence and the location of the helical and non-helical parts of the structure is suggested. The presence in the sequence of four critical groups; proline, aspartic acid, glutamic acid, or histidine appears to be necessary (although the last three are not sufficient) for a helical disruption to form. Additional support for this correlation is obtained from analyses of proline replacement in mutant and variant proteins. A mechanism based on hydrophobic bonding is proposed as a rationale for the apparent behavior of these groups. On the basis of these rules and correlations, secondary structures can be proposed for lysozyme and tobacco mosaic virus protein which are consistent with several pieces of evidence.     

Electrical circuitry in biology: emerging principles from protein structure

The rate of electron transfer by tunnelling decreases exponentially with distance, but is generally not rate limiting for distances up to 14A, enabling the robust design of redox systems. Fast transfer over distances greater than 14A is accomplished using diffusible electron carriers, an array of closely spaced redox centres or large-scale motion of a redox-active domain. Recent advances indicate that all three mechanisms are used in interprotein electron transfer. The classic stratagem, diffusible electron carriers, may be extended with either of the other designs. The use of an array of solvent-exposed, closely spaced redox centres can maximise productive collisions. Also, the use of conformational sampling via domain motion within the electron transfer complex optimises tunnelling probability.     

The limit of accuracy of protein modeling: influence of crystal packing on protein structure

The size of the protein database (PDB) makes it now feasible to arrive at statistical conclusions regarding structural effects of crystal packing. These effects are relevant for setting upper practical limits of accuracy on protein modeling. Proteins whose crystals have more than one molecule in the asymmetric unit or whose structures were determined at least twice by X-ray crystallography were paired and their differences analyzed. We demonstrate a clear influence of crystal environment on protein structure, including backbone conformations, hinge-like motions and side-chain conformations. The positions of surface water molecules tend to be variable in different crystal environments while those of ligands are not. Structures determined by independent groups vary more than structures determined by the same authors. The use of different refinement methods is a major source for this effect. Our pair-wise analysis derives a practical limit to the accuracy of protein modeling. For different crystal forms, the limit of accuracy (C(alpha), root-mean-square deviation (RMSD)) is approximately 0.8A for the entire protein, which includes approximately 0.3A due to crystal packing. For organized secondary elements, the upper limit of C(alpha) RMSD is 0.5-0.6A while for loops or protein surface it reaches 1.0A. Twenty percent of exposed side- chains exhibit different chi(1+2) conformations with approximately half of the effect also resulting from crystal packing. A web based tool for analysis and graphic presentation of surface areas of crystal contacts is available (http://ligin.weizmann.ac.il/cryco).     

Structural investigation of a phosphorylation-catalyzed, isoaspartate-free, protein succinimide: crystallographic structure of post-succinimide His15Asp histidine-containing protein

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through the high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.     

The central domain of the matrix protein of HIV-1: influence on protein structure and virus infectivity

The central region of the matrix protein p17 of HIV-1 is known to be essential during virus assembly. We substituted alanines for amino acid triplets in this region of p17 (amino acid residues 47 to 55: NPG LLE TSE). Introduction of the respective mutations into the gag-coding sequence of HI-proviruses and subsequent transfection into Cos-7 cells led to particle production and release. Exchange of LLE resulted in the production of non-infectious particles. These residues may be important for correct folding and assembly of the processed matrix protein and the production of infectious HIV. In vitro studies of wild-type and mutated matrix proteins using spectroscopic methods (NMR, fluorescence, CD) yielded detailed data about structure and stability. Two-dimensional NMR spectroscopy showed that wild-type and mutant proteins (p17-NPG and p17-TSE) are well folded. Besides structural changes at the mutated site, chemical shift changes indicate small but significant long range structural rearrangements. The stability against chemically and thermally induced unfolding of the mutants p17-NPG and p17-TSE was slightly decreased, while that of p17-LLE was drastically diminished. The alterations have only a local effect on protein folding for the mutants p17-NPG and p17-TSE, and the globular tertiary structure remains nearly unchanged. For p17-LLE, however, the substitutions seem to trigger significant changes in structural elements.     

The products from lipase-catalysed hydrolysis of bovine milkfat kill Helicobacter pylori in vitro

Free fatty acids and monoglycerides released from milkfat by partial pregastric lipase-catalysed hydrolysis are bactericidal towards Helicobacter pylori. Two milkfat preparations were investigated: a normal bovine milkfat, and a fractionated milkfat preparation, termed ModFat, enriched in triglycerides containing short- and medium-chain fatty acids. The released products were tested for bactericidal potency against H. pylori. The potencies of the respective preparations were consistent with expected potencies calculated from individual free fatty acid and monoglyceride concentrations and their lauric acid equivalence factors (Ki). ModFat products were more bactericidal, in accordance with release of free fatty acid types of high potency, and addition of the surfactant Tween 80 to the hydrolysed lipid increased potency eight times more than did addition of lecithin. Tween 80 micelles have smaller aggregation numbers, and the mixed micelles of Tween 80/free fatty acids would be more likely to expose the bacteria to higher apparent free fatty acid concentrations.     

Regulation of glycosylation. The influence of protein structure on N-linked oligosaccharide processing

The Sindbis virus glycoproteins, E1 and E2, comprise a useful model system for evaluating the effects of local protein structure on the processing of N-linked oligosaccharides by Golgi enzymes. The conversion of oligomannose to N-acetyllactosamine (complex) oligosaccharides is hindered to different extents at the four glycosylation sites, so that the complex/oligomannose ratio decreases in the order E1-Asn139 greater than E2-Asn196 greater than E1-Asn245 greater than E2-Asn318. The processing steps most susceptible to interference were deduced from the oligosaccharide compositions at hindered sites in virus from baby hamster kidney cells (BHK), chick embryo fibroblasts (CEF), and normal and hamster sarcoma virus (HSV)-transformed hamster fibroblasts (Nil-8). Persistence of Man6-9GlcNAc2 was taken to indicate interference with alpha 2-mannosidase(s) I (alpha-mannosidase I), Man5GlcNAc2, with UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase I (GlcNAc transferase I), and unbisected hybrid glycans, with GlcNAc transferase I-dependent alpha 3(alpha 6)-mannosidase (alpha-mannosidase II). Taken together, the results indicate that all four sites acquire a precursor oligosaccharide with equally high efficiency, but alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are all impeded at E2-Asn318 and, to a lesser extent, at E1-Asn245. In contrast, sialic acid and galactose transfer to hybrid glycans (in BHK cells) is virtually quantitative even at E2-Asn318. E2-Asn318 carried no complex oligosaccharides, but the structures of those at E1-Asn245 indicate almost complete GlcNAc transfer by UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase II (GlcNAc transferase II), galactosylation, and sialylation. Because the E2-Asn318 and E1-Asn245 glycans have previously been shown to be less accessible to a steric probe than those at E2-Asn196 or E1-Asn139, a simple explanation for these results would be that alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are more susceptible to steric hindrance than are the later processing steps examined. Finally, in addition to these site-specific effects, the overall extent of viral oligosaccharide processing varied with host and cellular growth status. For example, alpha-mannosidase I processing is more complete in BHK cells compared to CEF, and in confluent Nil-8 cells compared to subconfluent or HSV-transformed Nil-8 cells.     

The effects of glucosinolates and their hydrolysis products on microbial growth

Rapemeal, which contains potentially toxic compounds such as glucosinolates, was assessed as a substrate for the growth of micro-organisms. The effects of glucosinolates and their degradation products were tested on a range of industrially important microbial species. Sinigrin (2-propenyl glucosinolate) was found to be relatively innocuous to all of the organisms tested but its hydrolysis to yield isothiocyanates, thiocyanates and nitriles resulted in inhibition of growth. The initial inhibitory sinigrin concentration before its hydrolysis was found to be species-dependent with Bacillus subtilis being the most resistant (80 μg ml^-1) and Saccharomyces cerevisiae (40 μg ml^-1) the most sensitive. Three Gram-positive organisms tested were found to be more resistant to hydrolysis products than other micro-organisms. Similar results were observed with phenylisothiocyanate for which inhibition was found to be inhibitor and cell concentration-dependent. Addition of thioglucoside glucohydrolase during active growth of Escherichia coli in a sinigrin-containing liquid medium reduced the number of viable cells. Similar effects were also observed in rapemeal media in which growth inhibition was dependent on the glucosinolate content of the rapemeal.     

The emerging structure of the Agrobacterium T-DNA transfer complex

Single-stranded DNA-protein complex (T-complex) is proposed to mediate T-DNA transfer from Agrobacterium to plant cells. A novel model for transfer is presented which incorporates features of both bacterial conjugation and viral infection. Specific protein components of the T-complex, its ultrastructure and possible functions in the plant cell are discussed.     

The fate of the hydrolysis products of thalidomide in the pregnant rabbit

1. The fate in the pregnant New Zealand White rabbit of oral doses of four ¹⁴C-labelled hydrolysis products of thalidomide, namely α-(o-carboxybenzamido)-glutarimide, 2-phthalimidoglutaramic acid, 2-phthalimidoglutaric acid and 2-(o-carboxybenzamido)glutaramic acid, administered on the 192nd hour of pregnancy has been studied. 2. About 60–95% of the administered ¹⁴C of each compound appears in the urine in 58hr. and the remainder is found in the faeces and in the gut and its contents. 3. Radioactivity is present in the plasma, liver, kidney, brain, muscle, fat and embryo. 4. The ¹⁴C-labelled substances in the plasma and embryo consist of the unchanged compounds and their further hydrolysis products. 5. Since the above four thalidomide hydrolysis products are found in the rabbit conceptus together with their further hydrolysis products after their oral administration to the pregnant rabbit, it appears that the teratogenic activity of thalidomide is due to the compound itself rather than to one or more of its hydrolysis products.     

The influence of potential predators on the habitat preferenda of emerging brown trout

Much research has focused on the developmental behaviour of fish and it has been shown that their sensory and physical capabilities evolve very quickly during their early life. Thus, ontogenesis could influence fishes preferences for particular environmental factors. Little is known about the habitat preferences of trout during the post-emergence phase and it is not known if they correspond to the preference curves established by the PHABSIM method for the' alevin phase'. Here, the downstream movement and habitat preferences of young emerging trout were studied in a flume. In the absence of predators, alevins preferred a water depth of 20 to 30 cm and pebble rather than gravel substratum. When emergence occurred in an area with 1⁺ trout and sculpin, Cottus gobio , almost all the emergent trout remained cryptic. When visible, most of them were in the shallowest area (10cm depth) where their preference for pebble substratum was less marked. The presence of 1⁺ trout and sculpin increased the movement downstream of young trout by 20% without changing the general and diel patterns of catches. Their presence also reduced the initial growth of 0⁺ trout.