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1. Intensity correlation analysis of Raman spectra of concentrated Ficoll solutions

   https://doi.org/10.1117/12.2609947  

   Rivera-Lopez, Yahira M.; Salih, Mohamed A.; Boukari, Fatima; Barnett, Cleon M.; Boukari, Hacene (March 2022, Proc. SPIE 11944, Multiscale Imaging and Spectroscopy III)

   Maitland, Kristen C.; Roblyer, Darren M.; Campagnola, Paul J. (Ed.)

   The intracellular environment is crowded with diverse biomacrolecules (~80-400 mg/ml), likely affecting various biological processes such as protein folding, binding of small molecules, enzymatic activity, and pathological protein aggregation. As a model we have been using solutions of Ficoll, a highly branched polysaccharide, to mimic the environment. Besides its biomedical applications (e.g. blood separation), it has been used as a macromolecular crowder in studies of protein folding and stability, cell volume signaling, tissue engineering, and nanotransport. In this study, our goal is to identify and assess Raman spectral signatures associated with Ficoll molecules and Ficoll-Ficoll interactions for future investigations of crowding effects. In addition to the Raman peaks of water (~1640 cm-1 and ~3200 cm-1) and dissolved O2 (~1556 cm-1) and N2 (~2331 cm-1) we identified a distinct Raman peak (~2900 cm-1) in the 1500-3500 cm-1 wavenumber range, which is associated with Ficoll and CH and CH2 stretching modes. As the Ficoll concentration increases, the intensity of the Ficoll Raman peaks increases while the intensity of the water Raman peaks decreases, the latter likely due to reduction of water content. Further, we have applied the intensity correlation analysis (ICA) method to assess systematic changes of Raman spectra with Ficoll concentration (up to 1000 mg/ml). ICA indicates an overall linear trend over the full wavenumber range, but also shows closed loops that can be attributed to slight changes of the profiles of certain peaks. The results demonstrate ICA as a potential insightful tool for identifying Ficoll in chemical analysis of crowded biological samples. 

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2. Protein folding and surface interaction phase diagrams in vitro and in cells

   https://doi.org/10.1002/1873-3468.14058  

   Gruebele, Martin (March 2021, FEBS Letters)

   Protein stability is subject to environmental perturbations such as pressure and crowding, as well as sticking to other macromolecules and quinary structure. Thus, the environment inside and outside the cell plays a key role in how proteins fold, interact, and function on the scale from a few molecules to macroscopic ensembles. This review discusses three aspects of protein phase diagrams: first, the relevance of phase diagrams to protein folding and functionin vitroand in cells; next, how the evolution of protein surfaces impacts on interaction phase diagrams; and finally, how phase separation plays a role on much larger length‐scales than individual proteins or oligomers, when liquid phase‐separated regions form to assist protein function and cell homeostasis. 

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3. In-Cell Association of a Bioorthogonal Tubulin

   https://doi.org/10.1021/acs.biomac.3c01253  

   Wang, Yuhan; Unnikrishnan, Mahima; Ramsey, Brooke; El Andlosy, Driss; Keeley, Alex T.; Murphy, Catherine J.; Gruebele, Martin (February 2024, Biomacromolecules)

   Studies of proteins from one organism in another organism’s cells have shown that such exogenous proteins stick more, pointing toward coevolution of the cytoplasm and protein surface to minimize stickiness. Here we flip this question around by asking whether exogenous proteins can assemble efficiently into their target complexes in a non-native cytoplasm. We use as our model system the assembly of BtubA and BtubB from Prosthecobacter hosted in human U-2 OS cells. BtubA and B evolved from eukaryotic tubulins after horizontal gene transfer, but they have low surface sequence identity with the homologous human tubulins and do not respond to tubulin drugs such as nocodazole. In U-2 OS cells, BtubA and B assemble efficiently into dimers compared to in vitro, and the wild-type BtubA and B proteins subsequently are able to form microtubules as well. We find that generic crowding effects (Ficoll 70 in vitro) contribute significantly to efficient dimer assembly when compared to sticking interactions (U-2 OS cell lysate in vitro), consistent with the notion that a generic mechanism such as crowding can be effective at driving assembly of exogenous proteins, even when protein-cytoplasm quinary structure and sticking have been modified in a non-native cytoplasm. A simple Monte Carlo model of in vitro and in-cell interactions, treating BtubA and B as sticky dipoles in a matrix of sticky or nonsticky crowders, rationalizes all the experimental trends with two adjustable parameters and reveals nucleation as the likely mechanism for the time-scale separation between dimer- and tubule formation in-cell and in vitro. 

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4. In silico protein dynamics in the human cytoplasm: Partial folding, misfolding, fold switching, and non‐native interactions

   https://doi.org/10.1002/pro.4790  

   Russell, Premila P. Samuel; Rickard, Meredith M.; Boob, Mayank; Gruebele, Martin; Pogorelov, Taras V. (October 2023, Protein Science)

   Abstract We examine the influence of cellular interactions in all‐atom models of a section of theHomo sapienscytoplasm on the early folding events of the three‐helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30‐microsecond simulations. We did however capture the formation of all three helices in a compact native‐like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta‐sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild‐type sequence. This study demonstrates that even fast‐folding proteins can get stuck in non‐native states in the cell, making them useful models for protein–chaperone interactions and early stages of aggregate formation relevant to cellular disease. 

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5. Resolving the enthalpy of protein stabilization by macromolecular crowding

   https://doi.org/10.1002/pro.4573  

   Stewart, Claire J.; Olgenblum, Gil I.; Propst, Ashlee; Harries, Daniel; Pielak, Gary J. (February 2023, Protein Science)

   Abstract Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use¹⁹F nuclear magnetic resonance spectroscopy to follow the reversible, two‐state unfolding thermodynamics of the N‐terminal Src homology 3 domain of theDrosophilasignal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein–crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG–protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG–protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered. 

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