Styrene‐maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native‐like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α‐olefin‐maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene‐co‐maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I‐containing nanodiscs that retain an annulus of native lipids and a native‐like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine‐tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.
- NSF-PAR ID:
- 10354956
- Date Published:
- Journal Name:
- Polymer Chemistry
- Volume:
- 13
- Issue:
- 31
- ISSN:
- 1759-9954
- Page Range / eLocation ID:
- 4547 to 4556
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
more » « less
Abstract Styrene‐maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native‐like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α‐olefin‐maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene‐co‐maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I‐containing nanodiscs that retain an annulus of native lipids and a native‐like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine‐tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.
-
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
-
more » « less
Rationale Simple, affordable, and rapid methods for identifying the molecular weight (MW) distribution and macromolecular composition of polymeric materials are limited. Current tools require extensive solvent consumption, linear calibrations, and expensive consumables. A simple method for the determination of average MW (
M n,M w) and chain end groups is demonstrated for synthetic homopolymer standards using direct injection electrospray ionization‐mass spectrometry (ESI‐MS) and an open‐sourced charge deconvolution (CDC) algorithm.Methods Five homopolymer standards in the 1–7 kDa MW range were analyzed using direct‐injection ESI‐MS on a quadrupole/time‐of‐flight mass spectrometer. The samples investigated, viz. two poly(ethylene oxide) (PEO) and two poly(styrene sulfonic acid) (PSS) standards with narrow polydispersity and one poly(
d ,l ‐alanine) (pAla) standard with undefined polydispersity, were chosen to illustrate challenges with ESI‐MS quantitation. Using the UniDec program, weight average MWs (M w) obtained from the charge‐deconvoluted spectra were compared to the reportedM wdata of the standards from size exclusion chromatography (SEC) measurements.Results The MW data derived for the PSS, PEO, and pAla standards agreed well with the corresponding reported
M wor MW range values. The method was able to provide MW, degree of polymerization (DP), and polydispersity index (PDI) information for polymers with narrow (PSS, PEO) as well as broader (pAla) molecular weight distribution; this feature provides an advantage over MW analysis via matrix‐assisted laser desorption/ionization (MALDI) for ESI‐compatible materials. PSS standards differing in average MW by only a few repeat units could be confidently distinguished. Additionally, the oligomeric resolution observed for all samples studied unveiled chain‐end information not available through chromatographic analysis.Conclusions Overall, the free and easy‐to‐use UniDec CDC algorithm provides a simple, alternative method to measuring MW and DP for polymeric materials without high solvent consumption, expensive ionization sources, or calibration curves. Information about the masses of individual oligomers and the possibility to further characterize these oligomers using tandem mass spectrometry and/or ion mobility techniques constitutes additional benefits of this approach vis‐à‐vis traditional MW and PDI elucidation through SEC.
-
more » « less
Abstract The cellular glycocalyx and extracellular matrix are rich in glycoproteins and proteoglycans that play essential physical and biochemical roles in all life. Synthetic mimics of these natural bottlebrush polymers have wide applications in biomedicine, yet preparation has been challenged by their high grafting and glycosylation densities. Using one-pot dual-catalysis polymerization of glycan-bearing α-amino acid
N -carboxyanhydrides, we report grafting-from glycopolypeptide brushes. The materials are chemically and conformationally tunable where backbone and sidechain lengths were precisely altered, grafting density modulated up to 100%, and glycan density and identity tuned by monomer feed ratios. The glycobrushes are composed entirely of sugars and amino acids, are non-toxic to cells, and are degradable by natural proteases. Inspired by native lipid-anchored proteoglycans, cholesterol-modified glycobrushes were displayed on the surface of live human cells. Our materials overcome long-standing challenges in glycobrush polymer synthesis and offer new opportunities to examine glycan presentation and multivalency from chemically defined scaffolds.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D2PY00602B
