ABSTRACT
Natural molecular machines contain protein components that undergo motion relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein machinery from designed axle and rotor components with internal cyclic or dihedral symmetry. We find that the axle-rotor systems assemble in vitro and in vivo as designed. Using cryo-electron microscopy, we find that these systems populate conformationally variable relative orientations reflecting the symmetry of the coupled components and the computationally designed interface energy landscape. These mechanical systems with internal degrees of freedom are a step toward the design of genetically encodable nanomachines.
Subject(s)
Proteins , Cryoelectron Microscopy , Motion , Proteins/geneticsABSTRACT
The crystal structure of native chicken fibrinogen (320 kDa) complexed with two synthetic peptides has been determined at a resolution of 2.7 A. The structure provides the first atomic-resolution view of the polypeptide chain arrangement in the central domain where the two halves of the molecule are joined, as well as of a putative thrombin-binding site. The amino-terminal segments of the alpha and beta chains, including fibrinopeptides A and B, are not visible in electron density maps, however, and must be highly disordered. The alphaC domain is also very disordered. A residue by residue analysis of the coiled coils with regard to temperature factor shows a strong correlation between mobility and plasmin attack sites. It is concluded that structural flexibility is an inherent feature of fibrinogen that plays a key role in both its conversion to fibrin and its subsequent destruction by plasmin.
Subject(s)
Crystallography, X-Ray , Fibrinogen/chemistry , Amino Acid Sequence , Animals , Cattle , Chickens , Crystallization , Disulfides/chemistry , Fibrinogen/metabolism , Humans , Lampreys , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Thrombin/metabolismABSTRACT
The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor. The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase. In each case phylogenetic trees were constructed and the distances determined for all pairs. Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate.
Subject(s)
Eukaryotic Cells/physiology , Evolution, Molecular , Phylogeny , Proteins/physiology , Adenosine Triphosphatases/physiology , Amino Acyl-tRNA Synthetases/physiology , Aspartate Carbamoyltransferase/physiology , Bacterial Proteins/physiology , Carbamoyl-Phosphate Synthase (Ammonia)/physiology , Gene Duplication , Ornithine Carbamoyltransferase/physiology , Peptide Elongation Factors/physiology , Proteins/genetics , Signal Recognition Particle/physiologySubject(s)
Carrier Proteins/chemistry , Coenzyme A Ligases/chemistry , Membrane Proteins/chemistry , Membrane Transport Proteins , Amino Acid Sequence , Animals , Carrier Proteins/metabolism , Evolution, Molecular , Fatty Acid Transport Proteins , Fatty Acids/metabolism , Membrane Proteins/metabolism , Molecular Sequence Data , Sequence AlignmentABSTRACT
A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.