Very little is known about the distribution of functional DNA, RNA, and protein molecules in sequence space. The question of how the number and complexity of distinct solutions to a particular biochemical problem varies with activity is an important aspect of this general problem. Here we present a comparison of the structures and activities of eleven distinct GTP-binding RNAs (aptamers). By experimentally measuring the amount of information required to specify each optimal binding structure, we show that defining a structure capable of 10-fold tighter binding requires approximately 10 additional bits of information. This increase in information content is equivalent to specifying the identity of five additional nucleotide positions and corresponds to an ∼1000-fold decrease in abundance in a sample of random sequences. We observe a similar relationship between structural complexity and activity in a comparison of two catalytic RNAs (ribozyme ligases), raising the possibility of a general relationship between the complexity of RNA structures and their functional activity. Describing how information varies with activity in other heteropolymers, both biological and synthetic, may lead to an objective means of comparing their functional properties. This approach could be useful in predicting the functional utility of novel heteropolymers.

Despite its continued observational successes, there is a persistent (and growing) interest in extending cosmology beyond the standard model, ΛCDM. This is motivated by a range of apparently serious theoretical issues, involving such questions as the cosmological constant problem, the particle nature of dark matter, the validity of general relativity on large scales, the existence of anomalies in the CMB and on small scales, and the predictivity and testability of the inflationary paradigm. In this paper, we summarize the current status of ΛCDM as a physical theory, and review investigations into possible alternatives along a number of different lines, with a particular focus on highlighting the most promising directions. While the fundamental problems are proving reluctant to yield, the study of alternative cosmologies has led to considerable progress, with much more to come if hopes about forthcoming high-precision observations and new theoretical ideas are fulfilled.Keywords: cosmology -dark energy -cosmological constant problem -modified gravitydark matter -early universe Cosmology has been both blessed and cursed by the establishment of a standard model: ΛCDM. On the one hand, the model has turned out to be extremely predictive, explanatory, and observationally robust, providing us with a substantial understanding of the formation of large-scale structure, the state of the early Universe, and the cosmic abundance of different types of matter and energy. It has also survived an impressive battery of precision observational tests -anomalies are few and far between, and their significance is contentious where they do arise -and its predictions are continually being vindicated through the discovery of new effects (B-mode polarization [1] and lensing [2,3] of the cosmic microwave background (CMB), and the kinetic Sunyaev-Zel'dovich effect [4] being some recent examples). These are the hallmarks of a good and valuable physical theory.On the other hand, the model suffers from profound theoretical difficulties. The two largest contributions to the energy content at late times -cold dark matter (CDM) and the cosmological constant (Λ) -have entirely mysterious physical origins. CDM has so far evaded direct detection by laboratory experiments, and so the particle field responsible for it -presumably a manifestation of "beyond the standard model" particle physics -is unknown. Curious discrepancies also appear to exist between the predicted clustering properties of CDM on small scales and observations. The cosmological constant is even more puzzling, giving rise to quite simply the biggest problem in all of fundamental physics: the question of why Λ appears to take such an unnatural value [5,6,7]. Inflation, the theory of the very early Universe, has also been criticized for being fine-tuned and under-predictive [8], and appears to leave many problems either unsolved or fundamentally unresolvable. These problems are indicative of a crisis.From January 14th-17th 2015, we held a conference in Oslo, Norway to surve...

During the evolution of proteins the pressure to optimize biological activity is moderated by a need for efficient folding. For most proteins, this is accomplished through spontaneous folding to a thermodynamically stable and active native state. However, in the extracellular bacterial alpha-lytic protease (alphaLP) these two processes have become decoupled. The native state of alphaLP is thermodynamically unstable, and when denatured, requires millennia (t1/2 approximately 1,800 years) to refold. Folding is made possible by an attached folding catalyst, the pro-region, which is degraded on completion of folding, leaving alphaLP trapped in its native state by a large kinetic unfolding barrier (t1/2 approximately 1.2 years). alphaLP faces two very different folding landscapes: one in the presence of the pro-region controlling folding, and one in its absence restricting unfolding. Here we demonstrate that this separation of folding and unfolding pathways has removed constraints placed on the folding of thermodynamically stable proteins, and allowed the evolution of a native state having markedly reduced dynamic fluctuations. This, in turn, has led to a significant extension of the functional lifetime of alphaLP by the optimal suppression of proteolytic sensitivity.

Aptamers, RNA sequences that bind to target ligands, are typically isolated by in vitro selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to contain a centrally located stable stem-loop. We used an off-rate selection protocol designed to maximize the enrichment of high-affinity aptamers. The selection produced a surprisingly large number of distinct sequence motifs and secondary structures, including seven different aptamers with K ds ranging from 500 to 25 nanomolar. The engineered stem-loop was present in the three highest affinity aptamers, and in 12 of 13 independent isolates with a single consensus sequence, suggesting that its inclusion increased the abundance of high-affinity aptamers in the starting pool.

Bispecific antibodies and asymmetric Fc fusion proteins offer opportunities for important advances in therapeutics. Bivalent IgG depends upon in vivo dimerization of its heavy chains, mediated by homodimeric association of its C(H)3 domains. We have developed a heterodimeric Fc platform that supports the design of bispecific and asymmetric fusion proteins by devising strand-exchange engineered domain (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers that are composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEED C(H)3], that may be genetically linked to one or more fusion partners. This investigation reports on the generation of mono-Fab-Sb and Sb-IL2 monocytokine as models. They were expressed at high levels in NS/0 cells, purified on recombinant protein A resin and were well-behaved in solution. When administered intravenously to mice, Sb pharmacokinetics exhibited the long serum half-life extensions typical of comparable Fc-containing immunofusion and IgG1 controls.

Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin (¹⁰Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three ¹⁰Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the β strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these β strand interactions, indicating that these nonloop residues can expand the available binding footprint.

We report here the solution structure of omega-conotoxin GVIA, a peptide antagonist of the N-type neuronal voltage-sensitive calcium channel. The structure was determined using two-dimensional NMR in combination with distance geometry and restrained molecular dynamics. The full relaxation matrix analysis program MARDIGRAS was used to generate maximum and minimum distance restraints from the crosspeak intensities in NOESY spectra. The 187 restraints obtained were used in conjunction with 23 angle restraints from vicinal coupling constants as input for the structure calculations. The backbones of the best 21 structures match with an average pairwise RMSD of 0.58 A. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 18-21, and 24-27, making this the smallest published peptide structure to contain a triple-stranded beta-sheet. Conotoxins have been shown to be effective neuroprotective agents in animal models of brain ischemia. Our results should aid in the design of novel nonpeptide compounds with potential therapeutic utility.

To better understand the structural basis for the observed patterns in substrate specificity, the backbone dynamics of alpha-lytic protease have been investigated using 15N relaxation measurements. The enzyme was inhibited with the peptide boronic acid N-tert-butyloxycarbonyl-Ala-Pro-boroVal [Kettner, C. A., et al. (1988) Biochemistry 27, 7682], which mimics interactions occurring in the tetrahedral transition state or nearby intermediates, and the dynamics of the unbound and inhibited enzyme were compared. Arrayed 2-D NMR spectra were acquired to measure T1, T2, and steady-state ¿1H¿-15N NOE of >95% of the backbone amides in both protein samples. The overall rotational correlation time tauc was found to be 8.1 ns. Values of the spectral density function J(omega) at omega = 0, omegaN, and approximately omegaH were derived from the relaxation results using reduced spectral density mapping [Ishima, R., & Nagayama, K. (1995) Biochemistry 34, 3162]. The resultant spectral densities were interpreted to indicate regions of fast motion (nanosecond to picosecond) and of intermediate chemical exchange (millisecond to microsecond). The protein has 13 regions with increased motion on the fast time scale; these generally fall on exterior turns and loops and most correlate with regions of higher crystallographic B-factors. Several stretches of backbone undergo intermediate chemical exchange, indicating motion or other processes that cause temporal chemical shift changes. A comparison of spectral densities for both the free and inhibited enzymes revealed that inhibitor binding preferentially stabilizes regions undergoing chemical exchange (which predominate around the active site) and only minimally affect regions of rapid motion. Slow motions, suggestive of backbone plasticity, are observed in most of the binding pocket residues. This may point to a mechanism for the observed broad specificity of the enzyme. The significance of the observed dynamics for substrate binding and specificity is discussed.