High-Dimensional Mutant and Modular Thermodynamic Cycles, Molecular Switching, and Free Energy Transduction

Understanding how distinct parts of proteins produce coordinated behavior has driven and continues to drive advances in protein science and enzymology. However, despite consensus about the conceptual basis for allostery, the idiosyncratic nature of allosteric mechanisms resists general approaches. Computational methods can identify conformational transition states from structural changes, revealing common switching mechanisms that impose multistate behavior. Thermodynamic cycles use factorial perturbations to measure coupling energies between side chains in molecular switches that mediate shear during domain motion. Such cycles have now been complemented by modular cycles that measure energetic coupling between separable domains. For one model system, energetic coupling between domains has been shown to be quantitatively equivalent to that between dynamic side chains. Linkages between domain motion, switching residues, and catalysis make nucleoside triphosphate hydrolysis conditional on domain movement, confirming an essential yet neglected aspect of free energy transduction and suggesting the potential generality of these studies

Entropy, likelihood and phase determination

A key step in solving X-ray crystal structures is to provide phases for the experimentally observed X-ray reflection amplitudes so that they can be used as coefficients in a Fourier synthesis to obtain an electron-density map. The various ways of doing this for macromolecules all make use of phases from a known structure, either from heavy-atom positions in isomorphous structures or from a related macromolecule whose structure is known. These methods can fall short of providing unambiguous phase choices, leading to substantial delays in solving structures. Poor initial phases can also lead to mistaken interpretations that are hard to rectify because automatic refinement programs reinforce any 'model bias'

Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding

Genetic coding is generally thought to have required ribozymes whose functions were taken over by polypeptide aminoacyl-tRNA synthetases (aaRS). Two discoveries about aaRS and their interactions with tRNA substrates now furnish a unifying rationale for the opposite conclusion: that the key processes of the Central Dogma of molecular biology emerged simultaneously and naturally from simple origins in a peptide•RNA partnership, eliminating the epistemological utility of a prior RNA world. First, the two aaRS classes likely arose from opposite strands of the same ancestral gene, implying a simple genetic alphabet. The resulting inversion symmetries in aaRS structural biology would have stabilized the initial and subsequent differentiation of coding specificities, rapidly promoting diversity in the proteome. Second, amino acid physical chemistry maps onto tRNA identity elements, establishing reflexive, nanoenvironmental sensing in protein aaRS. Bootstrapping of increasingly detailed coding is thus intrinsic to polypeptide aaRS, but impossible in an RNA world. These notions underline the following concepts that contradict gradual replacement of ribozymal aaRS by polypeptide aaRS: (i) aaRS enzymes must be interdependent; (ii) reflexivity intrinsic to polypeptide aaRS production dynamics promotes bootstrapping; (iii) takeover of RNA-catalyzed aminoacylation by enzymes will necessarily degrade specificity; (iv) the Central Dogma's emergence is most probable when replication and translation error rates remain comparable. These characteristics are necessary and sufficient for the essentially de novo emergence of a coupled gene-replicase-translatase system of genetic coding that would have continuously preserved the functional meaning of genetically encoded protein genes whose phylogenetic relationships match those observed today

Augmenting the anisotropic network model with torsional potentials improves PATH performance, enabling detailed comparison with experimental rate data

PATH algorithms for identifying conformational transition states provide computational parameters-time to the transition state, conformational free energy differences, and transition state activation energies-for comparison to experimental data and can be carried out sufficiently rapidly to use in the high throughput mode. These advantages are especially useful for interpreting results from combinatorial mutagenesis experiments. This report updates the previously published algorithm with enhancements that improve correlations between PATH convergence parameters derived from virtual variant structures generated by RosettaBackrub and previously published kinetic data for a complete, four-way combinatorial mutagenesis of a conformational switch in Tryptophanyl-tRNA synthetase

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg2+ ion in a manner that allows it to assist in transition state stabilization. The Mg2+ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg2+·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg2+ coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg2+. Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS

Urzymology: Experimental Access to a Key Transition in the Appearance of Enzymes

Urzymes are catalysts derived from invariant cores of protein superfamilies. Urzymes from both aminoacyl-tRNA synthetase classes possess sophisticated catalytic mechanisms: pre-steady state bursts, significant transition-state stabilization of both amino acid activation, and tRNA acylation. However, they have insufficient specificity to ensure a fully developed genetic code, suggesting that they participated in synthesizing statistical proteins. They represent a robust experimental platform from which to articulate and test hypotheses both about their own ancestors and about how they, in turn, evolved into modern enzymes. They help reshape numerous paradigms from the RNA World hypothesis to protein structure databases and allostery

Before You Say Yes: A Planning Guide for Speakers

We need guidelines to help us decide whether to accept invitations to speak, whether to a class on campus or a special interest group outside. As educators and workshop organizers, we could also use suggestions on how to approach potential speakers. This article describes a single-page format that can be used to guide the planning process. Essential elements include contact information, location and organization of the activity, audience, learning goals, expected content, conclusions, and evaluation. Use of this planning sheet can give organization to an often haphazard process of planning, and enhance the potential of achieving the learning goals of a presentation

A modified PATH algorithm rapidly generates transition states comparable to those found by other well established algorithms

PATH rapidly computes a path and a transition state between crystal structures by minimizing the Onsager-Machlup action. It requires input parameters whose range of values can generate different transition-state structures that cannot be uniquely compared with those generated by other methods. We outline modifications to estimate these input parameters to circumvent these difficulties and validate the PATH transition states by showing consistency between transition-states derived by different algorithms for unrelated protein systems. Although functional protein conformational change trajectories are to a degree stochastic, they nonetheless pass through a well-defined transition state whose detailed structural properties can rapidly be identified using PATH