Binding of Dipeptides to Fatty Acid Membranes Explains Their Colocalization in Protocells but Does Not Select for Them Relative to Unjoined Amino Acids.

Dipeptides, which consist of two amino acids joined by a peptide bond, have been shown to have catalytic functions. This observation leads to fundamental questions relevant to the origin of life. How could peptides have become colocalized with the first protocells? Which structural features would have determined the association of amino acids and peptides with membranes? Could the association of dipeptides with protocell membranes have driven molecular evolution, favoring dipeptides over individual amino acids? Using pulsed-field gradient nuclear magnetic resonance, we find that several prebiotic amino acids and dipeptides bind to prebiotic membranes. For amino acids, the side chains and carboxylate contribute to the interaction. For dipeptides, the extent of binding is generally less than that of the constituent amino acids, implying that other mechanisms would be necessary to drive molecular evolution. Nevertheless, our results are consistent with a scheme in which the building blocks of the biological polymers colocalized with protocells prior to the emergence of RNA and proteins.

Studies of Dynamic Binding of Amino Acids to TiO2 Nanoparticle Surfaces by Solution NMR and Molecular Dynamics Simulations.

Adsorption of biomolecules onto material surfaces involves a potentially complex mechanism where molecular species interact to varying degrees with a heterogeneous material surface. Surface adsorption studies by atomic force microscopy, sum frequency generation spectroscopy, and solid-state NMR detect the structures and interactions of biomolecular species that are bound to material surfaces, which, in the absence of a solid-liquid interface, do not exchange rapidly between surface-bound forms and free molecular species in bulk solution. Solution NMR has the potential to complement these techniques by detecting and studying transiently bound biomolecules at the liquid-solid interface. Herein, we show that dark-state exchange saturation transfer (DEST) NMR experiments on gel-stabilized TiO2 nanoparticle (NP) samples detect several forms of biomolecular adsorption onto titanium(IV) oxide surfaces. Specifically, we use the DEST approach to study the interaction of amino acids arginine (Arg), lysine (Lys), leucine (Leu), alanine (Ala), and aspartic acid (Asp) with TiO2 rutile NP surfaces. Whereas Leu, Ala, and Asp display only a single weakly interacting form in the presence of TiO2 NPs, Arg and Lys displayed at least two distinct bound forms: a species that is surface bound and retains a degree of reorientational motion and a second more tightly bound form characterized by broadened DEST profiles upon the addition of TiO2 NPs. Molecular dynamics simulations indicate different surface bound states for both Lys and Arg depending on the degree of TiO2 surface hydroxylation but only a single bound state for Asp regardless of the degree of surface hydroxylation, in agreement with results obtained from the analysis of DEST profiles.

A Step toward Molecular Evolution of RNA: Ribose Binds to Prebiotic Fatty Acid Membranes, and Nucleosides Bind Better than Individual Bases Do.

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.

How internal cavities destabilize a protein.

Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å-3.

Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes.

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

Narrowing the gap between experimental and computational determination of methyl group dynamics in proteins.

Nuclear magnetic resonance (NMR) spin relaxation has become the mainstay technique to sample protein dynamics at atomic resolution, expanding its repertoire from backbone 15N to side-chain 2H probes. At the same time, molecular dynamics (MD) simulations have become increasingly powerful to study protein dynamics due to steady improvements of physical models, algorithms, and computational power. Good agreement between generalized Lipari-Szabo order parameters derived from experiment and MD simulation has been observed for the backbone dynamics of a number of proteins. However, the agreement for the more dynamic side-chains, as probed by methyl group relaxation, was much worse. Here, we use T4 lysozyme (T4L), a protein with moderate tumbling anisotropy, to showcase a number of improvements that reduce this gap by a combined evaluation of NMR relaxation experiments and MD simulations. By applying a protein force field with accurate methyl group rotation barriers in combination with a solvation model that yields correct protein rotational diffusion times, we find that properly accounting for anisotropic protein tumbling is an important factor to improve the match between NMR and MD in terms of methyl axis order parameters, spectral densities, and relaxation rates. The best agreement with the experimentally measured relaxation rates is obtained by a posteriori fitting the appropriate internal time correlation functions, truncated by anisotropic overall tumbling. In addition, MD simulations led us to account for a hitherto unrealized artifact in deuterium relaxation experiments arising from strong coupling for leucine residues in uniformly 13C-enriched proteins. For T4L, the improved analysis reduced the RMSD between MD and NMR derived methyl axis order parameters from 0.19 to 0.11. At the level of the spectral density functions, the improvements allow us to extract the most accurate parameters that describe protein side-chain dynamics. Further improvement is challenging not only due to force field and sampling limitations in MD, but also due to inherent limitations of the Lipari-Szabo model to capture complex dynamics.

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78➔Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.

Dynamics of dehaloperoxidase-hemoglobin A derived from NMR relaxation spectroscopy and molecular dynamics simulation.

Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHP's backbone helical regions and turns have average order parameters of S2 = 0.87 ±â€¯0.03 and S2 = 0.76 ±â€¯0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τm is 9.49 ±â€¯1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant Rex using the Lipari-Szabo model-free formalism. These include Lys3, Ile6, Leu13, Gln18, Arg32, Ser48, Met49, Thr56, Phe60, Arg69, Thr71 Cys73, Ala77, Asn81, Gly95, Arg109, Phe115, Leu127 and Met136, which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains >4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O2, H2O and H2O2 to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.

Nuclear magnetic resonance-based determination of dioxygen binding sites in protein cavities.

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O2 gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O2 in solvent to 8.9 mM, O2 -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O2 binding sites and their atomic coordinates from the 1/r6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O2 -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O2 revealed dioxygen ingress and egress as well as rotational and translational motions of O2 in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O2 association with the internal protein cavities.

Aberrant increase of NMR signal in hydrogen exchange experiments. Observation and explanation.

Hydrogen exchange (HX) NMR spectroscopy is widely used for monitoring structure, stability and dynamics of proteins at the level of individual residues. The stochastic replacement of protons by deuterons typically leads to an exponential decrease of the NMR signals. However, an unusual signal increase was observed in HX of several amides for T4 lysozyme L99A. This effect can be attributed to peak sharpening as a result of reduced dipolar relaxation from proximal amide protons that experience more rapid hydrogen/deuterium (H/D) exchange. The behavior was specifically observed at the termini of secondary structure elements, where large differences in protection against H/D exchange are observed. This effect is expected to be more widespread in NMR HX studies, and is important for the accurate determination of protection factors.

Detecting O2 binding sites in protein cavities.

Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in (1)H, (15)N, and (13)C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins.

Lysozyme as a pH-responsive valve for the controlled release of guest molecules from mesoporous silica.

Mesoporous silica nanoparticles show promise as a drug-carrier vehicle for biomedical applications, but the development of simple, biocompatible capping systems has remained a challenge. We have found that lysozyme molecules can act as a pH-responsive nanovalve to block and unlock the pore entrances of MCM-41 nanoparticles for guest molecules. Our experiments indicate that pore blocking is due to a pH-induced conformational change by which the effective size of the protein is changed in a reversible manner. This effect may form the basis of a controlled-release system without the need to functionalize the pore mouth and caps.

Assembling wormlike micelles in tubular nanopores by tuning surfactant-wall interactions.

Threadlike molecular assemblies are excluded from narrow pores unless attractive interactions with the confining pore walls compensate for the loss of configurational entropy. Here we show that wormlike surfactant micelles can be assembled in the 8 nm tubular nanopores of SBA-15 silica by adjusting the surfactant-pore-wall interactions. The modulation of the interactions was achieved by coadsorption of a surface modifier that also provides control over the partitioning of wormlike aggregates between the bulk solution and the pore space. We anticipate that the concept of tuning the interactions with the pore wall will be applicable to a wide variety of self-assembling molecules and pores.