Understanding and predicting how amino acid substitutions affect proteins is key to our basic understanding of protein function and evolution. Amino acid changes may affect protein function in a number of ways including direct perturbations of activity or indirect effects on protein folding and stability. We have analysed 6749 experimentally determined variant effects from multiplexed assays on abundance and activity in two proteins (NUDT15 and PTEN) to quantify these effects, and find that a third of the variants cause loss of function, and about half of loss-of-function variants also have low cellular abundance. We analyse the structural and mechanistic origins of loss of function, and use the experimental data to find residues important for enzymatic activity. We performed computational analyses of protein stability and evolutionary conservation and show how we may predict positions where variants cause loss of activity or abundance. In this way, our results link thermodynamic stability and evolutionary conservation to experimental studies of different properties of protein fitness landscapes.
We present a new software framework for Markov chain Monte Carlo sampling for simulation, prediction, and inference of protein structure. The software package contains implementations of recent advances in Monte Carlo methodology, such as efficient local updates and sampling from probabilistic models of local protein structure. These models form a probabilistic alternative to the widely used fragment and rotamer libraries. Combined with an easily extendible software architecture, this makes PHAISTOS well suited for Bayesian inference of protein structure from sequence and/or experimental data. Currently, two force-fields are available within the framework:PROFASI and OPLS-AA/L, the latter including the generalized Born surface area solvent model. A flexible command-line and configuration-file interface allows users quickly to set up simulations with the desired configuration. PHAISTOS is released under the GNU General Public License v3.0. Source code and documentation are freely available from http://phaistos.sourceforge.net. The software is implemented in C11 and has been tested on Linux and OSX platforms.
Understanding and predicting how amino acid substitutions affect proteins is key to practical uses of proteins, and to our basic understanding of protein function and evolution. Amino acid changes may affect protein function in a number of ways including direct perturbations of activity or indirect effects on protein folding and stability. We have analysed 6749 experimentally determined variant effects from multiplexed assays on abundance and activity in two proteins (NUDT15 and PTEN) to quantify these effects, and find that a third of the variants cause loss of function, and about half of loss-of-function variants also have low cellular abundance. We analyse the structural and mechanistic origins of loss of function, and use the experimental data to find residues important for enzymatic activity. We performed computational analyses of protein stability and evolutionary conservation and show how we may predict positions where variants cause loss of activity or abundance.
BackgroundAccurately covering the conformational space of amino acid side chains is essential for important applications such as protein design, docking and high resolution structure prediction. Today, the most common way to capture this conformational space is through rotamer libraries - discrete collections of side chain conformations derived from experimentally determined protein structures. The discretization can be exploited to efficiently search the conformational space. However, discretizing this naturally continuous space comes at the cost of losing detailed information that is crucial for certain applications. For example, rigorously combining rotamers with physical force fields is associated with numerous problems.ResultsIn this work we present BASILISK: a generative, probabilistic model of the conformational space of side chains that makes it possible to sample in continuous space. In addition, sampling can be conditional upon the protein's detailed backbone conformation, again in continuous space - without involving discretization.ConclusionsA careful analysis of the model and a comparison with various rotamer libraries indicates that the model forms an excellent, fully continuous model of side chain conformational space. We also illustrate how the model can be used for rigorous, unbiased sampling with a physical force field, and how it improves side chain prediction when used as a pseudo-energy term. In conclusion, BASILISK is an important step forward on the way to a rigorous probabilistic description of protein structure in continuous space and in atomic detail.
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