Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline

Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C(gamma)-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. C(gamma)-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides.In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the C(gamma)-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines.We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the C(gamma)-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms

An automatic assessment scheme for steel quality inspection

This paper presents an automatic system for steel quality assessment, by measuring textural properties of carbide distributions. In current steel inspection, specially etched and polished steel specimen surfaces are classified manually under a light microscope, by comparisons with a standard chart. This procedure is basically two-dimensional, reflecting the size of the carbide agglomerations and their directional distribution. To capture these textural properties in terms of image features, we first apply a rich set of image-processing operations, including mathematical morphology, multi-channel Gabor filtering, and the computation of texture measures with automatic scale selection in linear scale-space. Then, a feature selector is applied to a 40-dimensional feature space, and a classification scheme is defined, which on a sample set of more than 400 images has classification performance values comparable to those of human metallographers. Finally, a fully automatic inspection system is designed, which actively selects the most salient carbide structure on the specimen surface for subsequent classification. The feasibility of the overall approach for future use in the production process is demonstrated by a prototype system. It is also shown how the presented classification scheme allows for the definition of a new reference chart in terms of quantitative measures.QC 20100525</p

The tunicate Salpa thompsoni :Population and feeding dynamics under bloom versus non-bloom conditions

This paper presents an automatic system for steel quality assessment, by measuring textural properties of carbide distributions. In current steel inspection, specially etched and polished steel specimen surfaces are classified manually under a light microscope, by comparisons with a standard chart. This procedure is basically two-dimensional, reflecting the size of the carbide agglomerations and their directional distribution. To capture these textural properties in terms of image features, we first apply a rich set of image-processing operations, including mathematical morphology, multi-channel Gabor filtering, and the computation of texture measures with automatic scale selection in linear scale-space. Then, a feature selector is applied to a 40-dimensional feature space, and a classification scheme is defined, which on a sample set of more than 400 images has classification performance values comparable to those of human metallographers. Finally, a fully automatic inspection system is designed, which actively selects the most salient carbide structure on the specimen surface for subsequent classification. The feasibility of the overall approach for future use in the production process is demonstrated by a prototype system. It is also shown how the presented classification scheme allows for the definition of a new reference chart in terms of quantitative measures.QC 20100525</p

Biosens. Bioelectron.

The painting Nasturtium Landscape - enamel on plywood and recycled timber, 30cm x 30cm, exhibited in Flores et Fructus, 2017 at the Robin Gibson Gallery, was formed by amalgamating both pre-painted panels, and recycled timber which had an existing patina of brush strokes, into a ‘loose’ grid formation. The work did not utilise an aesthetic of overlapping, nor did it follow a strict grid for its configuration. The aesthetic outcome was however, indebted to a methodology of ‘fitting’ by which sections of the work had to be adjusted in shape to align with neighboring components. Given the vibrant orange colour of the top half of the work, and the long horizontal alignment of it to the bottom of the work, the notion, or illusion of a optical horizon can be seen to exist in the work, and thus the works title

In vitro synthesis of GPCRs in a solid supported planar lipid membrane system

Membrane protein research depends largely on the availability of functional protein in an instrumental set- up for application of biophysical investigations. We used an in vitro synthesis strategy in the presence of a solid supported lipid membrane as a new experimental strategy for the expression and membrane insertion of complex membrane proteins. Thus we realized the insertion of such a complex membrane protein, the odorant receptor OR5 from rat, which belongs to the highly important class of G-Protein coupled receptors (GPCRs). By applying a set of different biophysical and biochemical tools, e.g. surface plasmon enhanced fluorescence spectroscopy (SPFS), immunological detection (ELISA), control of integration based on enzymatic digestion, we proofed that membrane insertion of the protein is complete, proceeds in a vectorial manner and provides the odorant receptor in a biological active form, which recognizes the corresponding ligand molecule, the odour lilial. This experimental system of in vitro expressed and tBLM inserted OR5 was further used for single molecule measurements with image correlation microscopy, a combination of total internal reflection fluorescence (TIRF) and fluorescence correlation spectroscopy (FCS). The measurements focused on the visualization of odorant receptor molecules and clusters, respectively, as well as the determination of their lateral diffusion within the artificial membrane system. We show the fluorescently labeled antibodies tagging affinity labels of the individual OR5 proteins, inserted in the membrane system. High-resolution image correlation microscopy, i.e. the spatio-temporal auto-correlation of the membrane proteins, yields information about the spatial protein distribution (incorporation density) as well as the protein mobility (diffusion in the membrane)