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Effect of Osmolytes on the Conformational Behavior of a Macromolecule in Cytoplasm-like Crowded Environment: A Femtosecond Mid-IR Pump-Probe Spectroscopy Study Achintya Kundu, Pramod Kumar Verma, and Minhaeng Cho J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b03297 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018
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The Journal of Physical Chemistry Letters
Effect of Osmolytes on the Conformational Behavior of a Macromolecule in Cytoplasm-like Crowded Environment: A Femtosecond Mid-IR Pump-Probe Spectroscopy Study Achintya Kundu†,┴, Pramod Kumar Verma§,┴, and Minhaeng Cho†,‡,* †
Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Seoul 02841, Republic of Korea ‡
§
Department of Chemistry, Korea University, Seoul 02841, Republic of Korea.
Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi-221005, India
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ABSTRACT Osmolytes found endogenously in almost all living beings play an important role in regulating cell volume under harsh environment. Here, to address the longstanding questions about underlying mechanism of osmolyte effects, we use femtosecond mid-IR pump-probe spectroscopy with two different IR probes that are the OD stretching mode of HDO and the azido stretching mode of azido-derivatized poly(ethylene glycol) dimethyl ether (PEGDME). Our experimental results show that protecting osmolytes bind strongly with water molecules and dehydrate polymer surface, which results in promoting intramolecular interactions of the polymer. In contrast, urea behaves like water molecules without significantly disrupting water Hbonding network and favors extended and random-coil segments of the polymer chain by directly participating in solvation of the polymer. Our findings highlight the importance of direct interaction between urea and macromolecule, while protecting osmolytes indirectly affect macromolecule through enhancing water-osmolyte interaction under crowded environment, which is the case that is often encountered in real biological systems. TOC GRAPHICS
KEYWORDS. Osmolytes, water dynamics, denaturing osmolytes, protecting osmolyte, polymer, hydrogen bonding, femtosecond, vibrational spectroscopy, and orientational relaxation.
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The Journal of Physical Chemistry Letters
Almost all living organisms need to adapt to environmental stresses such as extreme pressure, temperature, and pH, cellular dehydration, high extracellular salt, and presence of denaturing highly-concentrated urea and salts inside cells like those in mammalian kidney.1-2 Cells adapt to such stresses by accumulating certain small organic molecules, known as osmolytes. Osmolytes are known to have profound effects on protein stability. A few of them are denaturants like urea, whereas others like trimethylglycine (TMG), trimethylamine N-oxide (TMAO) etc. act as protecting osmolytes even under denaturing conditions by stabilizing native protein structures. Such TMAO’s ability to promote protein folding was interestingly used to study the mechanisms of mis-folding processes of disease-causing proteins like prion protein,3 tau protein (related to Alzheimer’s disease),4 and α-synuclein.5-6 An important question that has not yet been fully elucidated is how osmolyte molecules affect protein stability in living cells? A number of studies emphasized the importance of either direct interactions between osmolyte molecules and protein via hydrogen bonds (H-bonds) and other electrostatic interactions or favorable van der Waals interactions between osmolyte molecules and protein’s amino acids, which essentially shift the equilibrium between folded and unfolded protein structure towards its folded (native) state.7-12 Other studies, however, showed that osmolyte molecules modulate or disrupt the H-bonding network of water, which then indirectly induces changes in the delicate balance between the forces and entropies of folded and unfolded protein states. As a result, the stability of the protein is modified.13-18 In the case of urea, recent time-resolved IR and vibrational sum-frequency-generation spectroscopic studies showed that urea does not make any long-range effect on the fluctuating Hbonding network and structural dynamics of water.8,
19-20
Only a small fraction of water
molecules that are doubly H-bonded to urea exhibit slow reorientation dynamics compared to
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those in bulk water. In contrast, TMAO, an important protecting osmolyte, interacts with water molecules strongly and has a tendency to slow down the rotation of water molecules close to its hydrophobic part.17,
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However, all the previous time-resolved vibrational spectroscopic
studies aimed at revealing osmolyte effects considered binary aqueous solutions consisting of water and osmolyte molecules without any other co-solute molecules. In a real biological system, osmolyte molecules are dissolved in complicated and crowded solution, e.g., intracellular environment, which contains various finite-sized molecules constituting 30-40 % of the total cell mass.24 Understanding the mechanism of osmolyte effects in such a crowded environment instead of simple osmolyte-water solutions is thus of significance in elucidating their biophysical roles. Nonetheless, since it is almost impossible to carry out time-resolved IR studies on osmolyte effects in vivo, we here consider aqueous osmolyte solutions with highly-concentrated macromolecules. Using two different IR probes that are deuterated water and azido-derivatized polymer in combination with fs mid-IR pump-probe (PP) method, we address two important questions: (i) what are osmolyte effects on fluctuating water H-bonding network structure as well as on vibrational population and rotational dynamics of water? and (ii) Do osmolyte molecules affect the conformation of macromolecule by directly interacting with the polymer or indirectly changing water H-bonding network? >> Scheme 1 > Figure 1
Figure 2 > Figure 3
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