Introduction of foreign peptides in surface loops of alkaline phosphatase

The introduction of foreign peptides into alkaline phosphatase that have a minimal influence on the enzymatic activity of a protein is described in the work. Calculated based on the methods of molecular dynamics the longest surface loops of alkaline phosphatase, which are not adjacent to both active center of a dimeric enzyme and contact surface between its monomers, were used as sites of the introduction of foreign peptides. The length of loops was estimated by several methods. Two loops were used to genetically engineer the introduction of peptides. Experiments demonstrated that the introduction of several peptides after the Ala218 residue of alkaline phosphatase insignificantly influences on the enzyme activity. According to experimental data, the selection of the loop for introducing the foreign peptide can be determined using the methods of molecular dynamics based on calculating the mobility of dihedral angles. In order to create macromolecules with new features by introducing foreign polypeptides in enzymatically active proteins, it is possible in practice to use an estimation of the loop length by means of the methods of molecular dynamics.     

Use of intensity-time studies as an aid to interpreting sweet taste chemoreception

The effects of temparature on the sweetness intensity and persistenceof some polyols are described. Also, the relative sweetnessof sucrose and D-glucose in isomole fractional sloutions isinvestigated and the effects of increasing concentration andvolume on the sweetness of these sugars is elucidated. Usingintensity–time profils, the apparent K_m values of 11 sweetenershave been deternined. These reflect the apparent binding affinitiesof receptors to sweeteners and correlate well with the thresholdconcentrations.     

Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

A successful HIV vaccine in addition to induction of antibody responses should elicit effective T cell responses. Here we described possible strategies for rational design of T-cell vaccine capable to induce high levels of both CD4+ and CD8+ T- cell responses. We developed artificial HIV-1 polyepitope T-cell immunogens based on the conserved natural CD8+ and CD4+ T cell epitopes from different HIV-1 strains and restricted by the most frequent major human leukocyte antigen (HLA) alleles. Designed immunogens contain optimized core polyepitope sequence and additional “signal” sequences which increase epitope processing and presentation to CD8+ and CD4+ T-lymphocytes: N-terminal ubiquitin, N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. As a result we engineered three T cell immunogens – TCI-N, TCI-N2, and TCI-N3, with different combinations of signal sequences. All designed immunogens were able to elicit HIV-specific CD4+ and CD8+ T cell responses following immunization. Attachment of either ubiquitin or ER-signal/LAMP-1 sequences increased both CD4+ and CD8+ mediated HIV-specific T cell responses in comparison with polyepitope immunogen without any additional signal sequences. Moreover, TCI-N3 polyepitope immunogen with ubiquitin generated highest magnitude of HIV-specific CD4+ and CD8+ T cell responses in our study. Obtained data suggests that attachment of signal sequences targeting polyepitope immunogens to either MHC class I or MHC class II presentation pathways may improve immunogenicity of T-cell vaccines. These results support the strategy of the rational T cell immunogen design and contribute to the development of effective HIV-1 vaccine.     

The spatial variability of methane emission from subtaiga and forest–steppe grass–moss fens of Western Siberia

Methane emission from the grass–moss fens of the Western Siberia subtaiga was studied using a static chamber method. It was established that CH₄ flux median ± half of the interquartile range in the studied wetland ecosystems constituted 4.9 ± 2.9 mg of CH₄/(m² h). It was shown that such a high spatial variability of emission is caused mainly by the difference in the water table level. It was found that, in these observations, a higher water table level correlates with lower emission values. The causes of this phenomenon are discussed, and recommendations for conducting field studies for estimating the regional flux are given.     

The interface of protein structure, protein biophysics, and molecular evolution

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction.     

A structure-activity study of thaumatin using pyridoxal 5'-phosphate (PLP) as a probe

The structural features responsible for the sensory propertiesof the sweet protein, thaumatin, have been investigated by sidechain modification of amino acid residues using pyridoxal 5'-phosphate(PLP). PLP molecules bind covalently to proteins by reactingwith the -amino group and the -amino group of lysine residues.Spectral and sensory studies have been performed on thaumatin-PLPderivatives prepared at various molar ratios. The incorporationof one mole of PLP into thaumatin causes substantial modificationof the sensory properties which include generation of astringency,an unpleasant taste and the loss of sweetness intensity. Theintroduction of more than one mole of PLP has no further effecton the gustatory properties of thaumatin. Removal by alkalinephosphatase of the phosphate group of PLP bound to thaumatinhas no influence on the ability of PLP to modify the sensorycharacteristics of thaumatin. This suggests that the sensoryalteration caused by PLP cannot be ascribed to the changes inthe net charge of the protein, but is likely to be due to themodification of specific lysine residue(s) which are thus implicatedin the sweet site.     

Biocomputing enters its adolescence

A report on the tenth Pacific Symposium on Biocomputing,Big Island, Hawaii, USA, 4-8 January 2005.