Concepts to Bolster Biorisk Management.

The rapid increase in the power of the life sciences has not been accompanied by a proportionate increase in the sophistication of biorisk management. Through conversations with thought leaders in biosafety and biosecurity, we have identified 19 concepts that are critical for biorisk management to continue to ensure the responsible and safe conduct of the life sciences in the future. Our work is not meant to be a comprehensive list, but rather a collection of topics that we hope will spark dialogue in the policy, research, and biorisk management communities.

Computational design of a modular protein sense-response system.

Sensing and responding to signals is a fundamental ability of living systems, but despite substantial progress in the computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here, we describe a generalizable computational strategy for designing sensor-actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation through split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site closely matches the design model. Our computational design strategy opens broad avenues to link biological outputs to new signals.

Basic Scholarship in Biosafety Is Critically Needed To Reduce Risk of Laboratory Accidents.

Our firm conducted a risk/benefit assessment of "gain-of-function" research, as part of the deliberative process following a U.S. moratorium on the research (U.S. Department of Health and Human Services, U.S. Government Gain-of-Function Deliberative Process and Research Funding Pause on Selected Gain-of-Function Research Involving Influenza, MERS, and SARS Viruses, 2014). Due to significant missing but theoretically acquirable data, our biosafety assessment faced limitations, and we were forced to provide a relative, instead of absolute, measure of risk (Gryphon Scientific, LLC, Risk and Benefit Analysis of Gain of Function Research, 2016). Here, we argue that many of these types of missing data represent large and stunning gaps in our knowledge of biosafety and argue that these missing data, once acquired via primary research efforts, would improve biosafety risk assessments and could be incorporated into biosafety practices to reduce risk of accidents. Governments invest billions in biological research; at least a small fraction of this support is warranted to prevent biological accidents.

Design of Light-Controlled Protein Conformations and Functions.

In recent years, interest in controlling protein function with light has increased. Light offers a number of unique advantages over other methods, including spatial and temporal control and high selectivity. Here, we describe a general protocol for engineering a protein to be controllable with light via reaction with an exogenously introduced photoisomerizable small molecule and illustrate our protocol with two examples from the literature: the engineering of the calcium affinity of the cell-cell adhesion protein cadherin, which is an example of a protein that switches from a native to a disrupted state (Ritterson et al. J Am Chem Soc (2013) 135:12516-12519), and the engineering of the opening and closing of the chaperonin Mm-cpn, an example of a switch between two functional states (Hoersch et al.: Nat Nanotechn (2013) 8:928-932). This protocol guides the user from considering which proteins may be most amenable to this type of engineering, to considerations of how and where to make the desired changes, to the assays required to test for functionality.

Design of a photoswitchable cadherin.

There is a growing interest in engineering proteins whose function can be controlled with the spatial and temporal precision of light. Here, we present a novel example of a functional light-triggered switch in the Ca-dependent cell-cell adhesion protein E-cadherin, created using a mechanism-based design strategy. We report an 18-fold change in apparent Ca(2+) binding affinity upon illumination. Our results include a detailed examination of functional switching via linked changes in Ca(2+) binding and cadherin dimerization. This design opens avenues toward controllable tools that could be applied to many long-standing questions about cadherin's biological function in cell-cell adhesion and downstream signaling.

A test on peptide stability of AMBER force fields with implicit solvation.

We used replica exchange molecular dynamics (REMD) simulations to evaluate four different AMBER force fields and three different implicit solvent models. Our aim was to determine if these physics-based models captured the correct secondary structures of two alpha-helical and two beta-peptides: the 14-mer EK helix of Baldwin and co-workers, the C-terminal helix of ribonuclease, the 16-mer C-terminal hairpin of protein G, and the trpzip2 miniprotein. The different models gave different results, but generally we found that AMBER ff96 plus the implicit solvent model of Onufriev, Bashford, and Case gave reasonable structures, and is fairly well-balanced between helix and sheet. We also observed differences in the strength of ion pairing in the solvent models, we but found that the native secondary structures were retained even when salt bridges were prevented in the conformational sampling. Overall, this work indicates that some of these all-atom physics-based force fields may be good starting points for protein folding and protein structure prediction.