Discovering pathways through ribozyme fitness landscapes using information theoretic quantification of epistasis.

The identification of catalytic RNAs is typically achieved through primarily experimental means. However, only a small fraction of sequence space can be analyzed even with high-throughput techniques. Methods to extrapolate from a limited data set to predict additional ribozyme sequences, particularly in a human-interpretable fashion, could be useful both for designing new functional RNAs and for generating greater understanding about a ribozyme fitness landscape. Using information theory, we express the effects of epistasis (i.e., deviations from additivity) on a ribozyme. This representation was incorporated into a simple model of the epistatic fitness landscape, which identified potentially exploitable combinations of mutations. We used this model to theoretically predict mutants of high activity for a self-aminoacylating ribozyme, identifying potentially active triple and quadruple mutants beyond the experimental data set of single and double mutants. The predictions were validated experimentally, with nine out of nine sequences being accurately predicted to have high activity. This set of sequences included mutants that form a previously unknown evolutionary "bridge" between two ribozyme families that share a common motif. Individual steps in the method could be examined, understood, and guided by a human, combining interpretability and performance in a simple model to predict ribozyme sequences by extrapolation.

Nanodisc Lipids Exhibit Singular Behaviors Implying Critical Phenomena.

Nanodiscs are broadly used for characterization of membrane proteins as they are generally assumed to provide a near-native environment. In fact, it is an open question whether the physical properties of lipids in nanodiscs and membrane vesicles of the same lipid composition are identical. Here, we investigate the properties of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and their mixtures) in two different sample types, nanodiscs and multilamellar vesicles, by means of spin-label electron spin resonance techniques. Our results provide a quantitative description of lipid dynamics and ordering, elucidating the molecular details of how lipids in the two sample types behave differently in response to temperature and lipid composition. We show that the properties of lipids are altered in nanodiscs such that the dissimilarity of the fluid and gel lipid phases is reduced, and the first-order phase transitions are largely abolished in nanodiscs. We unveil that the ensemble of lipids in the middle of a nanodisc bilayer, as probed by the end-chain spin-label 16-PC, is promoted to a state close to a miscibility critical point, thereby rendering the phase transitions continuous. Critical phenomena have recently been proposed to explain features of the heterogeneity in native cell membranes. Our results lay the groundwork for how to establish a near-native environment in nanodiscs with simple organization of lipid components.

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation.

Functional biomolecules, such as RNA, encapsulated inside a protocellular membrane are believed to have comprised a very early, critical stage in the evolution of life, since membrane vesicles allow selective permeability and create a unit of selection enabling cooperative phenotypes. The biophysical environment inside a protocell would differ fundamentally from bulk solution due to the microscopic confinement. However, the effect of the encapsulated environment on ribozyme evolution has not been previously studied experimentally. Here, we examine the effect of encapsulation inside model protocells on the self-aminoacylation activity of tens of thousands of RNA sequences using a high-throughput sequencing assay. We find that encapsulation of these ribozymes generally increases their activity, giving encapsulated sequences an advantage over nonencapsulated sequences in an amphiphile-rich environment. In addition, highly active ribozymes benefit disproportionately more from encapsulation. The asymmetry in fitness gain broadens the distribution of fitness in the system. Consistent with Fisher's fundamental theorem of natural selection, encapsulation therefore leads to faster adaptation when the RNAs are encapsulated inside a protocell during in vitro selection. Thus, protocells would not only provide a compartmentalization function but also promote activity and evolutionary adaptation during the origin of life.

Structural polymorphism and substrate promiscuity of a ribosome-associated molecular chaperone.

Trigger factor (TF) is a highly conserved multi-domain molecular chaperone that exerts its chaperone activity at the ribosomal tunnel exit from which newly synthesized nascent chains emerge. TF also displays promiscuous substrate binding for a large number of cytosolic proteins independent of ribosome binding. We asked how TF recognizes a variety of substrates while existing in a monomer-dimer equilibrium. Paramagnetic nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy were used to show that dimeric TF displays a high degree of structural polymorphism in solution. A series of peptides has been generated to quantify their TF binding affinities in relation with their sequence compositions. The results confirmed a previous predication that TF preferentially binds to peptide fragments that are rich in aromatic and positively charged amino acids. NMR paramagnetic relaxation enhancement analysis showed that TF utilizes multiple binding sites, located in the chaperone domain and part of the prolyl trans-cis isomerization domain, to interact with these peptides. Dimerization of TF effectively sequesters most of the substrate binding sites, which are expected to become accessible upon binding to the ribosome as a monomer. As TF lacks ATPase activity, which is commonly used to trigger conformational changes within molecular chaperones in action, the ribosome-binding-associated disassembly and conformational rearrangements may be the underlying regulatory mechanism of its chaperone activity.

Protocells.

Lai and Chen introduce the role of compartmentalization in the evolution of life.

Identifying Protein Conformational Dynamics Using Spin-label ESR.

Spin-label electron spin resonance (ESR) has emerged as a powerful tool to characterize protein dynamics. One recent advance is the development of ESR for resolving dynamical components that occur or coexist during a biological process. It has been applied to study the complex structural and dynamical aspects of membranes and proteins, such as conformational changes in protein during translocation from cytosol to membrane, conformational exchange between equilibria in response to protein-protein and protein-ligand interactions in either soluble or membrane environments, protein oligomerization, and temperature- or hydration-dependent protein dynamics. As these topics are challenging but urgent for understanding the function of a protein on the molecular level, the newly developed ESR methods to capture individual dynamical components, even in low-populated states, have become a great complement to other existing biophysical tools.

The role of cardiolipin in promoting the membrane pore-forming activity of BAX oligomers.

BCL-2-associated X (BAX) protein acts as a gatekeeper in regulating mitochondria-dependent apoptosis. Under cellular stress, BAX becomes activated and transforms into a lethal oligomer that causes mitochondrial outer membrane permeabilization (MOMP). Previous studies have identified several structural features of the membrane-associated BAX oligomer; they include the formation of the BH3-in-groove dimer, the collapse of the helical hairpin α5-α6, and the membrane insertion of α9 helix. However, it remains unclear as to the role of lipid environment in determining the conformation and the pore-forming activity of the BAX oligomers. Here we study molecular details of the membrane-associated BAX in various lipid environments using fluorescence and ESR techniques. We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers.

Coupling of lipid membrane elasticity and in-plane dynamics.

Biomembranes exhibit liquid and solid features concomitantly with their in-plane fluidity and elasticity tightly regulated by cells. Here, we present experimental evidence supporting the existence of the dynamics-elasticity correlations for lipid membranes and propose a mechanism involving molecular packing densities to explain them. This paper thereby unifies, at the molecular level, the aspects of the continuum mechanics long used to model the two membrane features. This ultimately may elucidate the universal physical principles governing the cellular phenomena involving biomembranes.

A Structurally Characterized Nonheme Cobalt-Hydroperoxo Complex Derived from Its Superoxo Intermediate via Hydrogen Atom Abstraction.

Bubbling O2 into a THF solution of CoII(BDPP) (1) at -90 °C generates an O2 adduct, Co(BDPP)(O2) (3). The resonance Raman and EPR investigations reveal that 3 contains a low spin cobalt(III) ion bound to a superoxo ligand. Significantly, at -90 °C, 3 can react with 2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOH) to form a structurally characterized cobalt(III)-hydroperoxo complex, CoIII(BDPP)(OOH) (4) and TEMPO•. Our findings show that cobalt(III)-superoxo species are capable of performing hydrogen atom abstraction processes. Such a stepwise O2-activating process helps to rationalize cobalt-catalyzed aerobic oxidations and sheds light on the possible mechanism of action for Co-bleomycin.

Crystal Structure Analysis of the Repair of Iron Centers Protein YtfE and Its Interaction with NO.

Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.

Solution Structure of Apoptotic BAX Oligomer: Oligomerization Likely Precedes Membrane Insertion.

Proapoptotic BAX protein is largely cytosolic in healthy cells, but it oligomerizes and translocates to mitochondria upon receiving apoptotic stimuli. A long-standing challenge has been the inability to capture any structural information beyond the onset of activation. Here, we present solution structures of an activated BAX oligomer by means of spectroscopic and scattering methods, providing details about the monomer-monomer interfaces in the oligomer and how the oligomer is assembled from homodimers. We show that this soluble oligomer undergoes a direct conversion into membrane-inserted oligomer, which has the ability of inducing apoptosis and structurally resembles a membrane-embedded oligomer formed from BAX monomers in lipid environment. Structural differences between the soluble and the membrane-inserted oligomers are manifested in the C-terminal helices. Our data suggest an alternative pathway of apoptosis in which BAX oligomer formation occurs prior to membrane insertion.

ESR study of interfacial hydration layers of polypeptides in water-filled nanochannels and in vitrified bulk solvents.

There is considerable evidence for the essential role of surface water in protein function and structure. However, it is unclear to what extent the hydration water and protein are coupled and interact with each other. Here, we show by ESR experiments (cw, DEER, ESEEM, and ESE techniques) with spin-labeling and nanoconfinement techniques that the vitrified hydration layers can be evidently recognized in the ESR spectra, providing nanoscale understanding for the biological interfacial water. Two peptides of different secondary structures and lengths are studied in vitrified bulk solvents and in water-filled nanochannels of different pore diameter (6.1~7.6 nm). The existence of surface hydration and bulk shells are demonstrated. Water in the immediate vicinity of the nitroxide label (within the van der Waals contacts, ~0.35 nm) at the water-peptide interface is verified to be non-crystalline at 50 K, and the water accessibility changes little with the nanochannel dimension. Nevertheless, this water accessibility for the nanochannel cases is only half the value for the bulk solvent, even though the peptide structures remain largely the same as those immersed in the bulk solvents. On the other hand, the hydration density in the range of ~2 nm from the nitroxide spin increases substantially with decreasing pore size, as the density for the largest pore size (7.6 nm) is comparable to that for the bulk solvent. The results demonstrate that while the peptides are confined but structurally unaltered in the nanochannels, their surrounding water exhibits density heterogeneity along the peptide surface normal. The causes and implications, especially those involving the interactions between the first hydration water and peptides, of these observations are discussed. Spin-label ESR techniques are proven useful for studying the structure and influences of interfacial hydration.

Intrinsic optical properties and divergent doping effects of manganese(II) on luminescence for tin(II) phosphite grown from a deep-eutectic solvent.

This is the first study on the ionothermal synthesis, intrinsic photoluminescence (PL), and dopant effects for tin(II) phosphite, a stereochemically active 5s(2) lone-pair-electron-containing compound, the fundamental properties of which have rarely been explored before. In a new deep-eutectic solvent, single-phased products of SnHPO(3) (1) and Sn(1-x)Mn(x)HPO(3) (2) have been achieved in high yield. The crystalline powder of 1 is nonenantiomorphic, with an intense second-harmonic generation comparable to that of potassium dihydrogen phosphate. Under UV excitation, it unexpectedly emits white PL, an important intrinsic property never discovered in tin(II) oxysalts. Electron paramagnetic resonance hyperfine splitting characteristic of manganese has been detected on 2 and a three-pulse electron-spin-echo envelope modulation technique implemented to locate its corresponding location in the inorganic host. On the basis of temperature-dependent PL and lifetime measurements, the incorporated Mn(2+) uncommonly acts as a sensitizer in enhancing white emission until extremely low temperatures, in which it would resume its normal role as an activator to give out characteristic orange light.

Mesopores provide an amorphous state suitable for studying biomolecular structures at cryogenic temperatures.

In nano-confinements, aqueous solutions can be found to remain in a liquid state at subfreezing temperatures. The finding provides a means of entering into previously inaccessible temperature regions for studying the dynamics and structure of bulk liquid. Here we show that studying biomolecular structures in nano-confinements improves the accuracy of cryostructures and provides better insight into the relationship between hydration water and biomolecules. Synthetic prion protein peptides are studied in two experimental conditions: (i) in confined nanochannels within mesoporous materials, and (ii) in vitrified bulk solvents, with a temperature range of 50-275 K, using cw/pulse ESR techniques. A large inhomogeneous lineshape broadening is only observed for the spectra from the vitrified bulk solvent below 70 K, suggesting a possible peptide clustering in the solution. The spin-counting and distance measurements by DEER-ESR provide further evidence that peptides are dispersed homogeneously in mesopores but heterogeneously in vitrified solvents wherein the biomolecular structure is disturbed due to heterogeneity in the bulk solvent structure. Our study demonstrates that the nanospace within mesoporous materials provides an amorphous environment that is better than vitrified bulk solvent for studying biostructures at cryogenic temperatures.