Bridging the physical scales in evolutionary biology: from protein sequence space to fitness of organisms and populations.

Bridging the gap between the molecular properties of proteins and organismal/population fitness is essential for understanding evolutionary processes. This task requires the integration of the several physical scales of biological organization, each defined by a distinct set of mechanisms and constraints, into a single unifying model. The molecular scale is dominated by the constraints imposed by the physico-chemical properties of proteins and their substrates, which give rise to trade-offs and epistatic (non-additive) effects of mutations.

Evolutionary dynamics of viral escape under antibodies stress: A biophysical model.

Viruses constantly face the selection pressure of antibodies, either from innate immune response of the host or from administered antibodies for treatment. We explore the interplay between the biophysical properties of viral proteins and the population and demographic variables in the viral escape. The demographic and population genetics aspect of the viral escape have been explored before; however one important assumption was the a priori distribution of fitness effects (DFE).

Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria.

Horizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10-90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.

Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics.

Unlabelled: Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene.

The influence of selection for protein stability on dN/dS estimations.

Understanding the relative contributions of various evolutionary processes-purifying selection, neutral drift, and adaptation-is fundamental to evolutionary biology. A common metric to distinguish these processes is the ratio of nonsynonymous to synonymous substitutions (i.e.

Merging molecular mechanism and evolution: theory and computation at the interface of biophysics and evolutionary population genetics.

The variation among sequences and structures in nature is both determined by physical laws and by evolutionary history. However, these two factors are traditionally investigated by disciplines with different emphasis and philosophy-molecular biophysics on one hand and evolutionary population genetics in another. Here, we review recent theoretical and computational approaches that address the crucial need to integrate these two disciplines.

Influenza A H1N1 pandemic strain evolution--divergence and the potential for antigenic drift variants.

The emergence of a novel A(H1N1) strain in 2009 was the first influenza pandemic of the genomic age, and unprecedented surveillance of the virus provides the opportunity to better understand the evolution of influenza. We examined changes in the nucleotide coding regions and the amino acid sequences of the hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) segments of the A(H1N1)pdm09 strain using publicly available data. We calculated the nucleotide and amino acid hamming distance from the vaccine strain A/California/07/2009 for each sequence.

Contribution of selection for protein folding stability in shaping the patterns of polymorphisms in coding regions.

The patterns of polymorphisms in genomes are imprints of the evolutionary forces at play in nature. In particular, polymorphisms have been extensively used to infer the fitness effects of mutations and their dynamics of fixation. However, the role and contribution of molecular biophysics to these observations remain unclear.

Positively selected sites in cetacean myoglobins contribute to protein stability.

Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8-1.

Highly abundant proteins favor more stable 3D structures in yeast.

To understand the variation of protein sequences in nature, we need to reckon with evolutionary constraints that are biophysical, cellular, and ecological. Here, we show that under the global selection against protein misfolding, there exists a scaling among protein folding stability, protein cellular abundance, and effective population size. The specific scaling implies that the several-orders-of-magnitude range of protein abundances in the cell should leave imprints on extant protein structures, a prediction that is supported by our structural analysis of the yeast proteome.

Protein quality control acts on folding intermediates to shape the effects of mutations on organismal fitness.

What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli's gene encoding dihydrofolate reductase (DHFR) and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: overexpression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains.

Protein biophysics explains why highly abundant proteins evolve slowly.

The consistent observation across all kingdoms of life that highly abundant proteins evolve slowly demonstrates that cellular abundance is a key determinant of protein evolutionary rate. However, other empirical findings, such as the broad distribution of evolutionary rates, suggest that additional variables determine the rate of protein evolution. Here, we report that under the global selection against the cytotoxic effects of misfolded proteins, folding stability (ΔG), simultaneous with abundance, is a causal variable of evolutionary rate.

The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for archaeal isopentenyl phosphate kinase.

Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK and H58 and K18 in Streptomyces wedmorensis FomA.

Structural basis for μ-opioid receptor binding and activation.

Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex.

A physical model reveals the mechanochemistry responsible for dynein's processive motion.

The molecular motor dynein is associated with various cellular activities, such as directed transport along microtubules and the rhythmic beating of the axoneme. Because of the size and complexity of the protein, a detailed understanding of the mechanochemistry that drives dynein's processive motion is lacking. To overcome this deficiency, we developed the first (to our knowledge) computational model for two-headed dynein that couples conformational changes of the motor's subunits to the biochemical steps involved in ATP hydrolysis.

Molecular modeling tools and approaches for CFTR and cystic fibrosis.

Cystic fibrosis is a multi-faceted disease resulting from the dysfunction of the CFTR channel. Understanding the structural basis of channel function and the structural origin of the defect is imperative in the development of therapeutic strategies. Here, we describe molecular modeling tools that, in conjunction with complementary experimental tools, lead to significant findings on CFTR channel function and on the effect of the pathogenic mutant F508del.

Multiscale approaches for studying energy transduction in dynein.

Cytoplasmic dynein is an important motor that drives all minus-end directed movement along microtubules. Dynein is a complex motor whose processive motion is driven by ATP-hydrolysis. Dynein's run length has been measured to be several millimetres with typical velocities in the order of a few nanometres per second.

A structural model of the pore-forming region of the skeletal muscle ryanodine receptor (RyR1).

Ryanodine receptors (RyRs) are ion channels that regulate muscle contraction by releasing calcium ions from intracellular stores into the cytoplasm. Mutations in skeletal muscle RyR (RyR1) give rise to congenital diseases such as central core disease. The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level.

Kinetic models for the coordinated stepping of cytoplasmic dynein.

To generate processive motion along a polymer track requires that motor proteins couple their ATP hydrolysis cycle with conformational changes in their structural subunits. Numerous experimental and theoretical efforts have been devoted to establishing how this chemomechanical coupling occurs. However, most processive motors function as dimers.

Identification and rational redesign of peptide ligands to CRIP1, a novel biomarker for cancers.

Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of cancers. Here we report on the use of phage display in combination with molecular modeling to identify a high-affinity ligand for CRIP1. Panning experiments using a circularized C7C phage library yielded several consensus sequences with modest binding affinities to purified CRIP1.

Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A.

Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding.

The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains.

Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its approximately 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites.

Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function.

Deletion of phenylalanine-508 (Phe-508) from the N-terminal nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family, disrupts both its folding and function and causes most cystic fibrosis. Most mutant nascent chains do not pass quality control in the ER, and those that do remain thermally unstable, only partially functional, and are rapidly endocytosed and degraded. Although the lack of the Phe-508 peptide backbone diminishes the NBD1 folding yield, the absence of the aromatic side chain is primarily responsible for defective CFTR assembly and channel gating.

Protein folding: then and now.

Over the past three decades the protein folding field has undergone monumental changes. Originally a purely academic question, how a protein folds has now become vital in understanding diseases and our abilities to rationally manipulate cellular life by engineering protein folding pathways. We review and contrast past and recent developments in the protein folding field.