Hydrophobic interactions drive the binding of
nonpolar ligands to the oily pockets of proteins and supramolecular
species in aqueous solution. As such, the wetting of host
pockets is expected to play a critical role in determining the
thermodynamics of guest binding. Here we use molecular
simulations to examine the impact of pressure on the wetting
and dewetting of the nonpolar pockets of a series of deep-cavity
cavitands in water. The portals to the cavitand pockets are
functionalized with both nonpolar (methyl) and polar (hydroxyl)
groups oriented pointing either upward or inward toward the
pocket. We find wetting of the pocket is favored by the hydroxyl
groups and dewetting is favored by the methyl groups. The
distribution of waters in the pocket is found to exhibit a two-state-like equilibrium between wet and dry states with a free energy
barrier between the two states. Moreover, we demonstrate that the pocket hydration of the cavitands can be collapsed onto a unified
adsorption isotherm by assuming the effective pressures within each cavitand pocket differ by a shift pressure that depends on the
chemical identity and number of functional groups placed about the portal. These observations support the development of a twostate
capillary evaporation model that accurately describes the equilibrium between states and naturally gives rise to the effective shift
pressures observed from simulation. This work demonstrates that the hydration of host pockets can be tuned following simple design
rules that in turn are expected to impact the thermodynamics of guest complexation.
more »
« less
Proximal charge effects on guest binding to a non-polar pocket
Science still does not have the ability to accurately predict the affinity that ligands have for proteins. In an attempt to address this, the Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) series of blind predictive challenges is a community-wide exercise aimed at advancing computational techniques as standard predictive tools in rational drug design. In each cycle, a range of biologically relevant systems of different levels of complexity are selected to test the latest modeling methods. As part of this on-going exercise, and as a step towards understanding the important factors in context dependent guest binding, we challenged the computational community to determine the affinity of a series of negatively and positively charged guests to two constitutionally isomeric cavitand hosts: octa-acid 1 , and exo -octa acid 2 . Our affinity determinations, combined with molecular dynamics simulations, reveal asymmetries in affinities between host–guest pairs that cannot alone be explained by simple coulombic interactions, but also point to the importance of host–water interactions. Our work reveals the key facets of molecular recognition in water, emphasizes where improvements need to be made in modelling, and shed light on the complex problem of ligand-protein binding in the aqueous realm.
more »
« less
- Award ID(s):
- 1805167
- NSF-PAR ID:
- 10157675
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 11
- Issue:
- 14
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 3656 to 3663
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Intracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular 'condensates' playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation/dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) and/or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA. Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations. Our lab has utilized complex coacervates assembled from oppositely-charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely-charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules. Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into the molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such ‘guest’ molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules such as viral vaccines. Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning from medicine to biocatalysis.more » « less
-
This article highlights the role of spatial confinement in controlling the fundamental behavior of molecules. Select examples illustrate the value of using space as a tool to control and understand excited state dynamics through a combination of ultrafast spectroscopy and conventional steady state methods. Molecules of interest were confined within a closed molecular capsule, derived from a cavitand known as octa acid (OA), whose internal void space is sufficient to accommodate molecules as long as tetracene and as wide as pyrene. The free space, i.e. the space that is left following the occupation of the guest within the host, is shown to play a significant role in altering the behavior of guest molecules in the excited state. The results reported here suggest that in addition to weak interactions that are commonly emphasized in supramolecular chemistry, the extent of empty space (i.e. the remaining void space within the capsule) is important in controlling the excited state behavior of confined molecules on ultrafast time scales. For example, the role of free space in controlling the excited state dynamics of guest molecules is highlighted by probing the cis-trans isomerization of stilbenes and azobenzenes within the OA capsule. Isomerization of both types of molecule are slowed when they are confined within a small space, with encapsulated azobenzenes taking a different reaction pathway compared to that in solution upon excitation to S¬2. In addition to steric constraints, confinement of reactive molecules in a small space helps to override the need for diffusion to bring the reactants together, thus enabling the measurement of processes that occur faster than the time scale for diffusion. The advantages of reducing free space and confining reactive molecules are illustrated by recording unprecedented excimer emission from anthracene and by measuring ultrafast electron transfer rates across the organic molecular wall. By monitoring the translational motion of anthracene pairs in a restricted space it has been possible to document the pathway undertaken by excited anthracene from inception to the formation of the excimer on the excited state surface. Similarly, ultrafast electron transfer experiments pursued here have established that the process is not hindered by a molecular wall. Apparently, the electron can cross the OA capsule wall provided the donor and acceptor are in close proximity. Measurements on the ultrafast time scale provide crucial insights for each of the examples presented here, emphasizing the value of both ‘space’ and ‘time’ in controlling and understanding the dynamics of excited molecules.more » « less
-
Exerting control on excited state processes has been a long-held goal in photochemistry. One approach to achieve control has been to mimic biological systems in Nature ( e.g. , photosynthesis) that has perfected it over millions of years by performing the reactions in highly organized assemblies such as membranes and proteins by restricting the freedom of reactants and directing them to pursue a select pathway. The duplication of this concept at a smaller scale in the laboratory involves the use of highly confined and organized assemblies as reaction containers. This article summarizes the studies in the author's laboratory using a synthetic, well-defined reaction container known as octa acid (OA). OA, unlike most commonly known cavitands, forms a capsule in water and remains closed during the lifetime of the excited states of included molecules. Thus, the described excited state chemistry occurs in a small space with hydrophobic characteristics. Examples where the photophysical and photochemical properties are dramatically altered, compared to that in organic solvents wherein the molecules are freely soluble, are presented to illustrate the value of a restricted environment in controlling the dynamics of molecules on an excited state surface. While the ground state complexation of the guest and host is controlled by well-known concepts of tight-fit, lock and key, complementarity, etc. , free space around the guest is necessary for it to be able to undergo structural transformations in the excited state, where the time is short. This article highlights the role of free space during the dynamics of molecules within a confined, inflexible reaction cavity.more » « less
-
more » « less
Summary Abscisic acid (
ABA ) receptors belong to theSTART domain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life.START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterizedSTART domain proteins are the 14PYR /PYL /RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently inNicotiana benthamiana coupled to untargetedLC ‐MS to identify candidate binding ligands. We optimized this method usingABA –PYL interactions and show thatABA co‐purifies with wild‐typePYL 5 but not a binding site mutant. TheK dofPYL 5 forABA is 1.1 μm , which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37START domain‐related proteins, which resulted in the identification of ligands that co‐purified withMLBP 1 (At4G01883) orMLP 165 (At1G35260). Metabolite identification and the use of authentic standards revealed thatMLBP 1 binds to monolinolenin, which we confirmed using recombinantMLBP 1. Monolinolenin also co‐purified withMLBP 1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C9SC06268H
