Superoxide Dismutase 1 Folding Stability as a Target for Molecular Tweezers in SOD1-Related Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation are hallmarks of many severe neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease. As a supramolecular ligand that binds to lysine and arginine residues, the molecular tweezer CLR01 was found to modify the aggregation pathway of disease-relevant proteins in vitro and in vivo with beneficial effects on toxicity. However, the molecular mechanisms of how tweezers exert these effects remain mainly unknown, hampering further drug development. Here, we investigate the modulation mechanism of unfolding and aggregation pathways of SOD1, which are involved in amyotrophic lateral sclerosis (ALS), by CLR01. Using a truncated version of the wildtype SOD1 protein, SOD1bar , we show that CLR01 acts on the first step of the aggregation pathway, the unfolding of the SOD1 monomer. CLR01 increases, by ∼10 °C, the melting temperatures of the A4V and G41D SOD1 mutants, which are commonly observed mutations in familial ALS. Molecular dynamics simulations and binding free energy calculations as well as native mass spectrometry and mutational studies allowed us to identify K61 and K92 as binding sites for the tweezers to mediate the stability increase. The data suggest that the modulation of SOD1 conformational stability is a promising target for future developments of supramolecular ligands against neurodegenerative diseases.

Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability.

Stress granules (SGs) are among the most studied membraneless organelles that form upon heat stress (HS) to sequester unfolded, misfolded, or aggregated protein, supporting protein quality control (PQC) clearance. The folding states that are primarily associated with SGs, as well as the function of the phase separated environment in adjusting the energy landscapes, remain unknown. Here, we investigate the association of superoxide dismutase 1 (SOD1) proteins with different folding stabilities and aggregation propensities with condensates in cells, in vitro and by simulation. We find that irrespective of aggregation the folding stability determines the association of SOD1 with SGs in cells. In vitro and in silico experiments however suggest that the increased flexibility of the unfolded state constitutes only a minor driving force to associate with the dynamic biomolecular network of the condensate. Specific protein-protein interactions in the cytoplasm in comparison to SGs determine the partitioning of folding states between the respective phases during HS.

Alteration of water absorption in the THz region traces the onset of fibrillation in proteins.

Using terahertz spectroscopy, we established the alteration of the collective hydration of water during the fibrillation process (native → intermediate → fibril) of a model protein bovine serum albumin. This label-free study concludes that water dynamics change systematically with protein conformational changes as it experiences a hydrophobic environment during the initial protein unfolding process, followed by the release of bound water during oligomerization and finally the hydrophobic interior of the fibril.

Polyethylene glycols affect electron transfer rate in phenosafranin-DNA complex.

Long distance electron transfer (ET) between small ligands and DNA is a much studied phenomenon and is principally believed to occur through electron (or hole) hopping. Several studies have been carried out in aqueous environments while in real biological milieu the DNA molecules experience a more dense and heterogeneous environment containing otherwise indifferent molecular crowders. It is therefore expected that the ET could get modified in the presence of crowding agent and to investigate that we have made elaborate studies on steady state and time-resolved (picosecond (ps) and femtosecond (fs)-resolved) emission properties of a phenosafranine (PSF) intercalated to calf thymus (CT) DNA in the presence of ethylene glycol (EG) and polyethylene glycols (PEG) of different chain lengths (PEG 200, 400 and 1000). The emission of PSF gets considerably quenched when intercalated to DNA; the quenching is released when PEGs are added into it. The structural integrity of the CT DNA has been established using circular dichroism spectroscopy. CD measurements have evidenced only marginal changes in the DNA structure upon the addition of PEGs. ps-Resolved fluorescence measurements show significant decrease in the contribution of the DNA induced quenched time-constant of PSF upon the addition of PEGs, however, fs-resolved measurements show less noticeable changes in the time constants. Our study shows that the electron hopping rate through the guanine base in DNA core remains unaffected whereas the 'through space' electron transfer process does get affected in the presence of molecular crowders.

Thermal stability modulation of the native and chemically-unfolded state of bovine serum albumin by amino acids.

Cells are crowded with various cosolutes including salts, osmolytes, nucleic acids, peptides and proteins. These cosolutes modulate the protein folding equilibrium in different ways, however, a unifying concept remains elusive. To elucidate the cosolute size-effect, macromolecular crowders are commonly compared to their monomeric building blocks (e.g. dextran vs. glucose or polyethylene glycol with different degrees of polymerization). To the best of our knowledge, such studies do not exist for protein crowders, raising the question of how single amino acids modulate the folding equilibrium. Therefore, we investigate the effect of glycine, alanine, proline and arginine on the stability of a model globular protein bovine serum albumin (BSA) upon thermal and urea-induced unfolding. We use three complementary techniques, fluorescence spectroscopy (as a local site-specific probe), circular dichroism (as a global probe for α-helical structure) and differential scanning calorimetry (to probe the energetics of unfolding). We find that the amino acids modulate BSA stability and unfolding, however, without following a particular trend with either the hydrophobicity scale or the solvent accessible surface area (SASA) of the added amino acids. Our data rather suggest that solvation effects play a role in understanding the cosolute effect.

Two Photon Spectroscopy Can Serve as a Marker of Protein Denaturation Pathway.

Rhodamine group of molecules are widely used dyes for imaging of biological molecules. Application of these dyes however includes a limitation that these molecules absorb in the visible range of the spectrum, which does not fall in the 'biologically transparent window' (BTW). Two photon absorption (TPA) process could come up with an alternate solution to this as these dyes could be excited in the near infrared (NIR) window to extract similar information. To validate this we have investigated TPA cross section (TPACS, σ2) of two rhodamine dyes, namely Rhodamine 6G (R6G), Rhodamine B (RhB), site selectively bound with a model protein, bovine serum albumin (BSA), by exciting at 800 nm. Two photon spectroscopy and imaging confirms the binding of the dye to the protein. The decreases in TPACS with increasing temperature at a fixed BSA concentration excellently follows the temperature induced structural transition of BSA as the protein transforms from a molten globule to unfolded conformation beyond 60 °C, which has previously been established through circular dichroism (CD) measurements. The thus established resemblance in TPACS and CD measurement trends thus strongly affirms the suitability of TPA process in protein imaging and as an alternative marker to tracking its conformational transformations using NIR radiation.

Soft interaction and excluded volume effect compete as polyethylene glycols modulate enzyme activity.

Polyethylene glycols (PEGs) can either preferentially bind to biomolecules or exert excluded volume effect depending upon their chain length and concentration. We have studied the effect of ethylene glycol (EG) and PEGs of different chain lengths (Mn 400 and 4000) on the enzyme efficiency of hen-egg-white lysozyme (HEWL) on Micrococcus lysodeikticus (M. Lys.) cell. The activity shows a bell-like profile as the turnover number increases from ~1.3 × 105 s-1 M-1 in water to ~1.7 × 105 s-1 M-1 in presence of 2% PEG-400 beyond which it decreases to ~0.7 × 105 s-1 M-1 at 20% PEG-400. Solvent polarity, excluded volume effect, soft nonspecific interactions and structural flexibility are found to be the competing factors which govern the overall enzyme activity as evidenced from circular dichroism (CD) and fluorescence measurements. Thermal unfolding temperature (Tm) of HEWL also shows a bell-shaped profile with PEG concentration which establishes possible correlation with its activity. We also observe a minimum in the activation energy barrier for the catalysis at low osmolyte concentrations. The maximum in the enzyme efficiency has been explained on the basis of an optimization between excluded volume effect and soft interaction among the protein and the cosolutes.

Effect of Phospholipid Headgroup Charge on the Structure and Dynamics of Water at the Membrane Interface: A Terahertz Spectroscopic Study.

Biological membranes are highly organized supramolecular assemblies of lipids and proteins. The membrane interface separates the outer (bulk) aqueous phase from the hydrophobic membrane interior. In this work, we have explored the microstructure and collective dynamics of the membrane interfacial hydration shell in zwitterionic and negatively charged phospholipid membrane bilayers using terahertz time-domain spectroscopy. We show here that the relaxation time constants of the water hydrogen bond network exhibit a unique "rise and dip" pattern with increasing lipid concentration. More importantly, we observed a dependence of the critical lipid concentration corresponding to the inflection point on the charge of the lipid headgroup, thereby implicating membrane electrostatics as a major factor in the microstructure and dynamics of water at the membrane interface. These results constitute one of the first experimental evidences of the modulation of the dielectric relaxation response of membrane interfacial water by membrane lipid composition in a concentration-dependent manner. Lipid-stringent membrane hydration could be relevant in the broader context of lipid diversity observed in biological membranes and the role of negatively charged lipids in membrane protein structure and function.

Decisive Role of Hydrophobicity on the Effect of Alkylammonium Chlorides on Protein Stability: A Terahertz Spectroscopic Finding.

Many biologically important processes involve a subtle interplay between Columbic and hydrophobic interactions among molecular groups with water. A comprehensive understanding of such processes, specially while occurring simultaneously in the same molecule is of practical importance. In this contribution, we report the ultrafast (subpicosecond to picosecond) collective hydrogen bond dynamics of water in the extended hydration layers in a series of alkylammonium chloride salts using THz time domain spectroscopic (TTDS) technique (0.3-1.6 THz (10-55 cm-1)). We found the THz absorption coefficient (α) of the salt solutions systematically vary with the salt type. We obtain the hydrogen bond relaxation dynamics by fitting the frequency dependent dielectric constants in a multiple Debye dielectric relaxation model. We found these salts to transform from being a water "structure breaker" to "structure maker" with increasing carbon content. We also investigate their effect on a model protein "bovine serum albumin" and found a systematic trend toward disrupting the protein secondary structure. The associated changes in the protein hydration in the presence of these salts have also been investigated using TTDS.

Collective hydration dynamics in some amino acid solutions: A combined GHz-THz spectroscopic study.

A detailed understanding of hydration of amino acids, the building units of protein, is a key step to realize the overall solvation processes in proteins. In the present contribution, we have made a combined GHz (0.2-50) to THz (0.3-2.0) experimental spectroscopic study to investigate the dynamics of water at room temperature in the presence of different amino acids (glycine, L-serine, L-lysine, L-tryptophan, L-arginine, and L-aspartic acid). The THz absorption coefficient, α(ν), of amino acids follows a trend defined by their solvent accessible surface area. The imaginary and real dielectric constants obtained in GHz and THz regions are fitted into multiple Debye model to obtain various relaxation times. The ∼100 ps time scale obtained in the GHz frequency region is attributed to the rotational motion of the amino acids. In the THz region, we obtain ∼8 ps and ∼200 fs time scales which are related to the cooperative dynamics of H-bond network and partial rotation or sudden jump of the under-coordinated water molecules. These time scales are found to be dependent on the amino acid type and the cooperative motion is found to be dependent on both the hydrophobic as well as the hydrophilic residue of amino acids.

Enhanced Catalytic Activity of α-Chymotrypsin in Cationic Surfactant Solutions: The Component Specificity Revisited.

Enhanced catalytic activity (super activity) of enzymes in the presence of surfactants is of key importance in "micellar enzymology"; such super activity is not very trivial, it is highly system specific, and the mechanism behind the activity enhancement is not always well apprehended. We report the catalytic activity of α-chymotrypsin (CHT) on ala-ala-phe-7-amido-4-methylcoumarin (AMC) in the presence of cationic surfactants of different hydrophobic chain lengths: dodecyltrimethylammonium bromide (DTAB), cetyltrimethylammonium bromide (CTAB) and octadecyltrimethylammonium bromide (OTAB). It is observed that in comparison to buffer the catalytic activity of CHT is enhanced 5-fold in premicellar DTAB solutions, while negligible changes are observed in CTAB and OTAB. Activity decreases considerably in the post micellar concentration, specifically for the latter two surfactants. A similar trend is also obtained in another substrate 2-napthyal acetate hydrolysis. Such surfactant specific superactivity is intriguing. The protein's secondary and tertiary structures in the presence of these surfactants are determined using circular dichroism (CD) spectroscopy and it is found that both CTAB and OTAB perturb the protein structure significantly, especially in the post micellar concentrations. DTAB, on the other hand, does not produce noticeable changes in the protein structure. The various pairwise interactions present in the system have been underlined using both steady-state and time-resolved fluorescence spectroscopy. Assuming a three-step kinetics model, we determine the free energy changes of the reaction, and the observations have been discussed in the light of the various interactions among the components.

Non-monotonic dynamics of water in its binary mixture with 1,2-dimethoxy ethane: A combined THz spectroscopic and MD simulation study.

A combined experimental (mid- and far-infrared FTIR spectroscopy and THz time domain spectroscopy (TTDS) (0.3-1.6 THz)) and molecular dynamics (MD) simulation technique are used to understand the evolution of the structure and dynamics of water in its binary mixture with 1,2-dimethoxy ethane (DME) over the entire concentration range. The cooperative hydrogen bond dynamics of water obtained from Debye relaxation of TTDS data reveals a non-monotonous behaviour in which the collective dynamics is much faster in the low Xw region (where Xw is the mole fraction of water in the mixture), whereas in Xw ∼ 0.8 region, the dynamics gets slower than that of pure water. The concentration dependence of the reorientation times of water, calculated from the MD simulations, also captures this non-monotonous character. The MD simulation trajectories reveal presence of large amplitude angular jumps, which dominate the orientational relaxation. We rationalize the non-monotonous, concentration dependent orientational dynamics by identifying two different physical mechanisms which operate at high and low water concentration regimes.

Effect of Short Chain Poly(ethylene glycol)s on the Hydration Structure and Dynamics around Human Serum Albumin.

We report the changes in the hydration dynamics around a globular protein, human serum albumin (HSA), in the presence of two short chain crowding agents, namely poly(ethylene glycol)s (PEG 200 and 400). The change in the network water structure is investigated using FTIR spectroscopy in the far-infrared (FIR) frequency range. Site specific changes are obtained by time-resolved fluorescence spectroscopic technique using the intrinsic fluorophore tryptophan (Trp214) of HSA. The collective hydration dynamics of HSA in the presence of PEG molecules are obtained using terahertz (THz) time domain spectroscopy (TTDS) and high intensity p-Ge THz measurements. Our study affirms a considerable perturbation of HSA hydration beyond a critical concentration of PEG.

Micelle induced dissociation of DNA-ligand complexes: The effect of ligand binding specificity.

We investigate the SDS micelle induced dissociation of a small fluorescent ligand 4',6-diamidino-2-phenylindole (DAPI) bound to DNAs of varying sequences. Steady state and time resolved fluorescence measurements affirm minor groove binding of DAPI to poly(dA).poly(dT) and calf thymus DNA while it intercalates in poly(dG).poly(dC). Calorimetric measurements identify the former mode to be entropy driven and the intercalation to be enthalpy driven. Addition of SDS micelles extracts the ligand out of the DNA and relocates it into the micelle independent of the DNA-ligand binding mode. This process is found to be endothermic which is compensated by a huge gain in the entropy. Circular dichroism measurements indicate that the micelles do not affect the structure of DNAs, however, binding and un-binding of DAPI can introduce noticeable alteration in the DNA structure and consequently on the associated hydration which is reflected in solvation measurement. Consideration of a simple two step equilibrium model seems inadequate to account for the observed thermodynamic costs in the dissociation process. The results have been discussed on the basis of an intricate enthalpy-entropy balance.

Does urea alter the collective hydrogen-bond dynamics in water? A dielectric relaxation study in the terahertz-frequency region.

We report the ultrafast collective hydrogen-bond dynamics of water in the extended hydration layer of urea by using terahertz time-domain spectroscopy in the frequency region of 0.3-2.0 THz. The complex dielectric function has been fitted using a Debye relaxation model, and the timescales obtained are in the order of approximately 9 ps and 200 fs for bulk water; this exhibits a considerable acceleration beyond the 4 M urea concentration and indicates a possible disruption in the collective hydrogen-bonded water-network structure, which, in turn, provides an indirect support for the water "structure-breaking" ability of urea. With 5 M urea in the presence of different concentrations of trimethylamine-N-oxide (TMAO), it was found that these parameters essentially follow the trend observed for TMAO itself, which signifies that any possible disruption of the water structure by urea is outdone by the strong hydrogen-bonding ability of TMAO, which explains its ability to revive urea-denatured proteins to their respective native states.

Collective hydration dynamics of guanidinium chloride solutions and its possible role in protein denaturation: a terahertz spectroscopic study.

The remarkable ability of guanidinium chloride (GdmCl) to denature proteins is a well studied yet controversial phenomenon; the exact molecular mechanism is still debatable, especially the role of hydration dynamics, which has been paid less attention. In the present contribution, we have addressed the issue of whether the collective hydrogen bond dynamics of water gets perturbed in the presence of GdmCl and its possible impact on the denaturation of a globular protein human serum albumin (HSA), using terahertz (THz) time domain spectroscopy (TTDS) in the frequency range of 0.3-2.0 THz. The collective hydrogen bond dynamics is determined by fitting the obtained complex dielectric response in a multiple Debye relaxation model. To compare the results, the studies were extended to two more salts: tetramethylguanidinium chloride (TMGdmCl) and sodium chloride (NaCl). It was concluded that the change in hydration dynamics plays a definite role in the protein denaturation process.

Short chain polyethylene glycols unusually assist thermal unfolding of human serum albumin.

In the present study we have investigated the thermal stability of the globular transport protein human serum albumin (HSA), in the presence of two small chain polyethylene glycols (namely PEG 200 and PEG 400). Both near- and far-UV circular dichroism (CD) study reveal that addition of PEG moderately increases the α-helical content of the protein without abruptly changing its tertiary structure. The hydration structure at the protein surface experiences a notable change at 30% PEG (v/v) concentration as evidenced from compressibility and dynamic light scattering (DLS) measurements. Thermal denaturation of HSA in the presence of PEG has been studied by CD and fluorescence spectroscopy using the intrinsic fluorophore tryptophan and it has been found that addition of PEG makes the protein more prone towards unfolding, which is in contrary to what has been observed in case of larger molecular weight polymers. The energetics of the thermal unfolding process has been obtained using differential scanning calorimetry (DSC) measurements. Our study concludes that both the indirect excluded volume principle as well as interaction of the polymer at the protein surface is responsible for the observed change of the unfolding process.