Project Summary/Abstract An important and currently unsolved challenge in evolutionary systems biology is to describe how the mechanisms and interactions between different levels of biological organization shape the evolution of proteins, cellular networks, and phenotypes. Our laboratory has been interested in understanding the inter-relationships between evolution and systems and cell biology for more than a decade. We have previously carried out computational and experimental analyses exploring evolutionary processes at multiple levels of organization. In this proposal we will integrate constraints and effects at the protein, network, cellular, and phenotypic scales, towards developing an integrated and quantitative framework describing protein evolution. We will use a combined computational-experimental approach to pursue such fundamental scientific questions as: How do global protein fitness landscapes affect short- and long-term patterns of protein evolution? How do the structure and function of cellular networks affect protein evolutionary changes? What is the role of cell type- and tissue-specific constraints on protein evolution? How does the evolution of protein repertoires influence the observed patterns and rates of phenotypic evolution? Because biological systems not only constrain, but are also themselves the products of evolution, exploring the aforementioned topics will also allow us to address important biological questions. For example, how and to what extent protein expression levels and the efficiency of protein function are optimized in evolution, and how changes in cellular networks may drive microbial phenotypic evolution. In addition to understanding fundamental biological questions, proposal results will have important implications for biomedicine. Specifically, they will allow to better understand and predict the quantitative effects of genetic mutations on protein function, cellular networks, diverse cell types and developmental stages, and ultimately on species’ phenotypes.