Lessons from Biomineralisation: the Role of Post-translational Modification

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

Lessons from Biomineralisation: the Role of Post-translational Modification

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

Ultra Low-cost DNA Miniprep Spin-Columns for Rapid Filter-Trapped Proteomics Sample Preparation

Complete proteolytic digestion in the preparation of proteins for bottom-up proteomic analysis is substantially improved by the use of detergents for complete denaturation. This however is incompatible with many proteases, and highly detrimental to LC-MS/MS data collection. Recently, filter-based methods such as FASP (Filter-Aided Sample Prep) have seen wide use due to their ability to remove detergents and other harmful reagents prior to digestion and mass spectrometric analysis. Unfortunately, these techniques can be variable and time consuming. Suspension trapping (S-Trapping) is a newer method that utilizes a depth-filter to trap flocculated proteins, and has proven to be a faster approach for proteomic analysis. Sample preparation by these methods requires careful control of protein concentrations in order to flocculate the sample for collection, and the cost of commercial solutions can be high. We hypothesized that protein suspensions also retain on silica-based filters due to ionic interactions mediated by the presence of sodium (Na+), SO42- and PO43-. As such, we sought to investigate if very low-cost DNA purification spin-filters, so called ‘minipreps’ could efficiently and reproducibly trap proteins for digest and LC-MS/MS analysis. Using model proteins and whole-cell lysates we compared digestion efficiencies, capacities, recovery and identification rates from samples prepared using DNA-minipreps and FASP-based protocols. Samples were analyzed using nano uHPLC MS-MS/MS and Label-Free-Quantitative (LFQ) proteomics. DNA-filters show low variability, excellent recovery, sensitivity, and proteome depth from a commercially obtainable device which costs < $0.25 (US) per sample.

Phenalenyls as tunable excellent molecular conductors and switchable spin filters

Phenalenyl-based radicals are stable radicals whose electronic properties can be tuned readily by heteroatom substitution. We employ density functional theory-based non-equilibrium Green’s function (NEGF-DFT) calculations to show that this class...

A high-spin diradical dianion and its bridged chemically switchable single-molecule magnet

Triplet diradicals have attracted tremendous attention due to their promising application in organic spintronics, organic magnets and spin filters. However, very few examples of triplet diradicals with singlet-triplet energy gaps...