We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. commonly used bioorthogonal motifs for Diels‐Alder cycloaddition with inverse electron demand (DA inv ). Here we present a highly efficient technology for protein functionalization with. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. GFP-display will be a helpful technique for many kinds of gene or protein studies related to amino acid substitutions such as the random mutagenesis in a gene of interest.īioorthogonal chemistry holds great potential to generate difficult‐to‐access protein‐protein conjugate architectures. the fluorescence intensity of GFP-fused polypeptide tends to increase with the average hydrophilicity of the fused polypeptide segment. the mobility shift is estimated to a certain extent using a new parameter called the 'GD value' calculated from the isoelectric point, hydrophilicity, and number of fused amino acids, and (iii). since the binding of dodecyl sulfate ions to acidic or hydrophilic amino acids is weaker than that to basic or hydrophobic amino acids, the ions bound weakly to the fused polypeptide segment are forced to come off by high concentrations of urea prior to the ions bound strongly, resulting in the mobility shift, (ii). An easy detection method for a single amino acid change based on this observation was called 'GFP-display.' Using various target polypeptides, staphylococcal protein A (SpA), Ras, p53, and human beta3 adrenergic receptor (AR), and their mobility-shift patterns resulting from the single amino acid changes, several important properties of GFP-display were revealed as follows: (i). The migrating position of green fluorescent protein (GFP)-fused polypeptide varied on an SDS/urea gel by a single amino acid change in the fused polypeptide segment. The fact that the three bound structures are essentially identical ensures productive interaction with the oligomeric membrane proteins, which are distinct for transport and chemotaxis.

Changes in the subtle contours of the binding site allow binding of maltodextrins of varying length with similarly high affinities. Hinge-bending between the two domains enables the participation of both domains in the binding and sequestering of the oligosaccharides. The polar groups and nonpolar patches are heavily involved in forming hydrogen bonds and van der Waals contacts, respectively, with complimentary residues in the groove. The bound maltooligosaccharides have a ribbon-like structure: the edges of the ribbon are occupied by polar hydroxyl groups and the flat surfaces are composed of nonpolar patches of the sugar ring faces.

MBP has a two-domain structure joined by a hinge-bending region which contains the substrate-binding groove. In addition, the structures demonstrate the different conformations that can be adopted by the oligosaccharide within the complex. Insights are provided into the structural plasticity of the protein, the binding affinity and the binding specificity with respect to alpha/beta anomeric preference and oligosaccharide length. The structures reveal differences between buried and surface binding sites and show the importance of hydrogen bonds and van der Waals interactions, especially those resulting from aromatic residue stacking. These structures provide details at the atomic level of many features of oligosaccharide binding. The structures of MBP complexed with maltose, maltotriose and maltotetraose have been refined to high resolutions (1.67 to 1.8 A). In order to gain new understanding of the function of MBP, especially its versatility in binding different linear and cyclic oligosaccharides with similar affinities, we have undertaken high-resolution X-ray analysis of three oligosaccharide-bound structures. The interaction between MBP and specific cytoplasmic membrane proteins initiates either active transport or chemotaxis. MBP also participates in chemotaxis towards maltooligosaccharides. Maltodextrin/maltose-binding protein (MBP M(r) = 40k) is a receptor protein which serves as an initial high-affinity binding component of the active-transport system of maltooligosaccharides in bacteria. Active-transport processes perform a vital function in the life of a cell, maintaining cell homeostasis and allowing access of nutrients.

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