A novel framework for engineering protein loops exploring length and compositional variation

Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum β-lactamases with loops of modified length and composition—areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

Topological principles of protein folding

Native topology correlates with folding rate: entangled topological relationships between protein loops facilitate folding. High numbers of topologically independent units (circuits) – normalized by size – are associated with fast folding kinetics.

Redesigning OmpA Loops Using Canonical Outer Membrane Protein Loop Structures

ABSTRACTProtein loops can be difficult to design and predict. There have been multiple different algorithms developed to predict the structure of loops. Outer membrane proteins are all beta barrels and these barrels have a variety of well-documented loop conformations. Here we test three different algorithms to predict the structure of outer membrane protein loops. We find the PETALS algorithm is superior for this purpose. We then experimentally test the effect of replacing the long loops of outer membrane protein OmpA with twelve shorter designed loops. Though we succeeded in creating the smallest known outer membrane barrel, we find that the designed loops do not have a strong effect on OmpA folding.