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Todays Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrows products and processes. On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the worlds grand challenges.

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.

Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site., QC 20150427

The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

Biomembranes are involved in some of the most basic cellular processes. These include ion transport, energy transduction, nerve transmission, sensory detection and cellular communication. The design of a new class of materials based on biomembrane components holds promise in diverse areas including optical recording media, chemical sensors, nanometer lithography, energy transducers and enzyme catalysis. However, future progress in these areas will depend on the development of new methods for elucidating the molecular basis for biomembrane function, self-assembly into higher order structures and modifying membrane components for biomaterial applications. In this project, we proposed to address these problems by developing powerful new methods based on molecular genetics and advanced biophysical techniques which have the capability to modify and characterize biomembranes on a molecular level. Key among these techniques will be site directed non-native amino acid replacement (SNAAR) along with the related technique of site directed isotope labeling (SDIL). These approaches when combined with FTIR spectroscopy will provide a powerful method for determining the role of individual amino acid residues in the functioning of a protein and in self-assembly as well as providing a new dimension in protein engineering, enabling the replacement of native amino acid residues with custom designed residues.