Organizational Unit:
Center for the Study of Systems Biology

Permanent Link
https://hdl.handle.net/1853/70795
Research Organization Registry ID
Description
Previous Names
Parent Organization
Parent Organization
Organizational Unit
Includes Organization(s)
ArchiveSpace Name Record

Filters
2011 - 2013 2
2
2
2
Reset filters

Settings
search.filters.applied.f.resourcesubtype: Lecture × Author: Shakhnovich, Eugene ×

Publication Search Results

Now showing 1 - 2 of 2
  • No Thumbnail Available
    Item
    Mapping Protein Folding on Organismal Fitness One Mutation at a Time
    (Georgia Institute of Technology, 2013-03-12) Shakhnovich, Eugene
    In this presentation I will describe our efforts at understanding how molecular properties of proteins determine fitness landscape of populations of carrier organisms. Recent multi-scale evolutionary models, which assume certain relationship between organismal fitness and stability of their proteins, have been successful in predicting such biological phenomena as lethal mutagenesis (six mutations per genome per generation), distributions of protein stabilities (‘’marginal’’ protein stability being a consequence of a mutation-selection balance), correlation between evolutionary rates and abundances. However, many of the underlying assumptions of these models have not been tested experimentally. Our recent efforts aim to close this gap. We explore fitness landscape of E.coli through controlled rational mutational genomic perturbations of expression level and stability of essential protein Dihydrofolate Reductase (DHFR). To that end we created transgenic E.coli, which carry specified mutations in the folA gene encoding DHFR and also placed the folA gene under an IPTG controllable promoter, making it possible to change the intracellular abundance of DHFR in a wide range. Using competition essays, we measured how biological fitness depends on Biophysical properties of DHFR such as its abundance in the cytoplasm, stability of its native state and folding intermediate, and catalytic activity. Mutant DHFR proteins in a few strains aggregated rendering them nonviable but the majority exhibited fitness higher than wild type at a growth temperature of 42oC. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced by soluble oligomerization that restores their structural stability and protects from aggregation. Further, we found that protein homeostasis plays a defining role in sculpting fitness effect of mutations. In particular, overexpression of GroEL as well as deletion of one of the proteases, Lon, resulted in complete recovery of fitness of unviable strains. Further study, including in vitro essays of ANS binding showed that GroEL and Lon compete for folding intermediate of DHFR and their relative concentrations determines the outcome. We developed a computational model to analyze this competition, which lead us to the conclusion that our observations cannot be reconciled with GroEL role as just caging device to protect DHFR mutants from aggregation and proteolysis. Rather, it must play an active role converting intermediate to folded molecules.
  • No Thumbnail Available
    Item
    Evolution: from atoms to organisms
    (Georgia Institute of Technology, 2011-02-15) Shakhnovich, Eugene
    Modern Biology is deeply rooted in Darwinian principles of mutations and selection. Population Genetics aims to address the effects of mutations and selection on populations in a quantitative way within the basic paradigm of ‘’fitness landscape’’, which postulates how genotypicchanges affect phenotype (e.g., fitness or growth rate of an organism or a tissue). No clear connection between the fitness effects of mutations and their effect on molecular properties of proteins has been systematically established – proteins are still treated as ‘’black boxes’’ in most population and organism level studies of evolution. In this talk I will present a systematic theoretical and experimental effort in our lab to go beyond this paradigm by developing multiscale models which relate molecular properties of proteins (their folding, function and interactions) to fitness of carrier organisms. By analyzing such models (both in simulations and analytically) we derived distribution of proteins stabilities which is very close to experimentally observed ones and predict its dependence of mutation rates and population size, linking ecology and molecular biophysics. Further we predicted and found correlation between protein stabilities and their abundances in cells as well as between protein abundances and strength of their interactions with other proteins. We discovered a universal physics-based speed limit on mutation rates in all organisms – ~6 missense mutations per essential part of the genome per replication. Further, we systematically experimentally probe fitness landscape by making controllable biophysical changes in proteins (varying stability and folding rates by mutations and abundances by manipulating upstream regions) with subsequent incorporation of mutant genes into E.coli chromosome and evaluating fitness of mutant strains in competition with wild-type. Our experiments confirm basic features of physics-based fitness landscape and add important new insights on how to make them more comprehensive and accurate.