110
S. Roy et al.
5.5 Osmolyte and Denaturant at Air-Water Interface
Osmolyte and denaturant are two opposite class of molecules. Osmolyte protects
cellular integrity and native protein structures by maintaining cell volume, against
changes of ambient conditions e.g. temperature, pressure, extracellular osmotic
concentration etc. [60, 61]. On the other hand, denaturant destabilizes native structure of proteins and destroy cellular integrity. Trimethylamine-N-oxide (TMAO,
(CH 3 ) 3 N
+ –O
− ) is one such well known organic osmolyte, while an alcohol of analogous chemical structure such as tert-butanol (TBA; (CH 3 ) 3 C–OH) is a denaturant
[62]. It is largely believed that unfavorable interaction of TMAO with hydrophobic
protein surface lead to its expulsion from the protein surface such that the protein
surface is exposed to the water that are strongly H-bonded with the expelled-TMAO.
The strongly H-bonded water at the protein surface favors the native (folded) structure of a protein, which has lower hydrophobic surface area exposed to water than
that of the corresponding denatured (unfolded) state [63–65]. Denaturants, on the
other hand, interact favorably with the hydrophobic protein surface and promotes
the protein to increase its hydrophobic surface area, leading to the denaturation. As
a result, the folding/unfolding of protein by osmolyte/denaturant is a phenomenon
at aqueous interface, which requires molecular level understanding.
As discussed in the Sect. 5.3, the negative CH stretch bands (CH 3
FR
= 2935 cm
−1
for TBA and CH 3
SS
= 2975 cm
−1 for TMAO; Fig. 12) show that both TBA and TMAO
are preferentially oriented as ‘methyl-up’ at the air-water interface. The H-bonded OH
stretch region (3000–3600 cm
−1 ) remains largely unperturbed in presence of TBA
(0.1 M); however, the amplitude of the dangling OH band (∼3710 cm
−1 ) decreases
from that of the neat air–water interface (compare red and black curves in Fig. 12a).
As dangling OH is a characteristic feature of topmost water, a reduction in its amplitude signifies dominant effect of TBA on the topmost water layer due its adsorption
on water surface. In the case of TMAO (3.0 M), the amplitude of the dangling OH is
largely unperturbed (compare red and black curve in Fig. 12b), suggesting that the
topmost water layer is largely unaffected by TMAO, even when present at significantly higher concentration than that of TBA (0.1 M) in the bulk water. Interestingly,
in the H-bonded OH stretch region, the Imχ
(2) signal is enhanced around 3250 cm
−1 .
An enhancement in the red region of the OH stretch spectrum is due to strong Hbonding of interfacial water that are associated with TMAO. Altogether, the spectral
features in the CH and OH stretch regions suggest preferential accumulation of TBA
on water surface while TMAO is depleted from the top surface but do reside in the
interfacial region (just below the top surface) while both of them exhibit preference
for the methyl-up orientation (Fig. 12c, d). Following the fact that air is hydrophobic,
the air-water interface can be considered as a proxy to the hydrophobic protein-water
interface. In that sense, preferential accumulation of TBA at the air-water interface
suggests the propensity of TBA to be adsorbed at hydrophobic protein surface. TMAO
on the other hand, is expected to be repelled from the protein surface. Such expulsion/adsorption of osmolyte/denaturant is fundamental to the folding/unfolding of
proteins in water.
S. Roy et al.
5.5 Osmolyte and Denaturant at Air-Water Interface
Osmolyte and denaturant are two opposite class of molecules. Osmolyte protects
cellular integrity and native protein structures by maintaining cell volume, against
changes of ambient conditions e.g. temperature, pressure, extracellular osmotic
concentration etc. [60, 61]. On the other hand, denaturant destabilizes native structure of proteins and destroy cellular integrity. Trimethylamine-N-oxide (TMAO,
(CH 3 ) 3 N
+ –O
− ) is one such well known organic osmolyte, while an alcohol of analogous chemical structure such as tert-butanol (TBA; (CH 3 ) 3 C–OH) is a denaturant
[62]. It is largely believed that unfavorable interaction of TMAO with hydrophobic
protein surface lead to its expulsion from the protein surface such that the protein
surface is exposed to the water that are strongly H-bonded with the expelled-TMAO.
The strongly H-bonded water at the protein surface favors the native (folded) structure of a protein, which has lower hydrophobic surface area exposed to water than
that of the corresponding denatured (unfolded) state [63–65]. Denaturants, on the
other hand, interact favorably with the hydrophobic protein surface and promotes
the protein to increase its hydrophobic surface area, leading to the denaturation. As
a result, the folding/unfolding of protein by osmolyte/denaturant is a phenomenon
at aqueous interface, which requires molecular level understanding.
As discussed in the Sect. 5.3, the negative CH stretch bands (CH 3
FR
= 2935 cm
−1
for TBA and CH 3
SS
= 2975 cm
−1 for TMAO; Fig. 12) show that both TBA and TMAO
are preferentially oriented as ‘methyl-up’ at the air-water interface. The H-bonded OH
stretch region (3000–3600 cm
−1 ) remains largely unperturbed in presence of TBA
(0.1 M); however, the amplitude of the dangling OH band (∼3710 cm
−1 ) decreases
from that of the neat air–water interface (compare red and black curves in Fig. 12a).
As dangling OH is a characteristic feature of topmost water, a reduction in its amplitude signifies dominant effect of TBA on the topmost water layer due its adsorption
on water surface. In the case of TMAO (3.0 M), the amplitude of the dangling OH is
largely unperturbed (compare red and black curve in Fig. 12b), suggesting that the
topmost water layer is largely unaffected by TMAO, even when present at significantly higher concentration than that of TBA (0.1 M) in the bulk water. Interestingly,
in the H-bonded OH stretch region, the Imχ
(2) signal is enhanced around 3250 cm
−1 .
An enhancement in the red region of the OH stretch spectrum is due to strong Hbonding of interfacial water that are associated with TMAO. Altogether, the spectral
features in the CH and OH stretch regions suggest preferential accumulation of TBA
on water surface while TMAO is depleted from the top surface but do reside in the
interfacial region (just below the top surface) while both of them exhibit preference
for the methyl-up orientation (Fig. 12c, d). Following the fact that air is hydrophobic,
the air-water interface can be considered as a proxy to the hydrophobic protein-water
interface. In that sense, preferential accumulation of TBA at the air-water interface
suggests the propensity of TBA to be adsorbed at hydrophobic protein surface. TMAO
on the other hand, is expected to be repelled from the protein surface. Such expulsion/adsorption of osmolyte/denaturant is fundamental to the folding/unfolding of
proteins in water.