Work done by me

Introduction and literature review

1.3 Work done by me

I undertook some basic and applied problems of protein science concerned with the

non-native aspects of macromolecule.

First, I tried to investigate the local or residual structures in different denatured proteins. Fluorescence from the indole side chain in trp(s) was applied as a spectroscopic probe to detect structural and rotational dynamics surrounding the trp(s) residue in denatured proteins.

Conformational heterogeneity in denatured protein poses a greatest challenge to locate the residual structures. Conventional methods like circular dichroism, infrared spectroscopy are not effective in highlighting their presence. However, NMR is widely technique to locate these structures. I therefore tried to use less complicated and time saving technique to hunt residual structures in different denatured proteins.

Two different biophysical parameters namely, bimolecular fluorescence quenching rate constant (kq) and steady-state anisotropy (rss) were used to monitor extent of exposure and rotational dynamics of trp(s) in a series of ten proteins subsequent to their overnight incubation in 6 M GdnCl at room temperature. Among ten proteins used in our investigation eight proteins namely, barstar, subtilisin carlsberg (SC), human serum albumin (HSA), melittin, myelin basic protein (MBP), glucagon, Ribonuclease T1 (RNase T1) and Trp-Met-Asp-Phe were having only one trp per polypeptide chain whereas bovine serum albumin (BSA) and hen egg white lysozyme (HEWL) were multi-tryptophan proteins. Bimolecular quenching constant (kq) reflects the efficiency of quencher or the accessibility of the fluorophores to the quencher, whereas steady-state anisotropy (rss) on the other hand reveals the extent of rotational freedom and dynamics of fluorophore at their lifetime scale. Since rss

depends upon both the lifetime and rotational correlation time of fluorophore

1

ss o

r r

τ θ

=

+

1.1

[where ro, which is a constant is the initial anisotropy (anisotropy observed in the absence of other depolarizing processes like rotational diffusion or energy transfer),

τ

is fluorescence lifetime and

θ

is rotational correlation time of fluorophore], change inrss value can be attributed by change in either

τ

_or

θ

_.

We monitored surface accessibility of trp(s) in presence of extrinsic quencher iodide (KI) after overnight incubation of proteins in 6 M GdnCl at room temperature.

Reduced

k_q

was observed in five proteins RNase T

1

, BSA, melittin, barstar and HEWL as compared to model compound N-Acetyl-L-tryptophanamide (NATA).

However, reduction in k

q

was more pronounced in RNase T

1

. Restricted rotational motion of trp(s) in same five proteins was also evident from increased r

ss

value after nightlong incubation in denaturant. On the contrary, there was no change in emission maximum of the proteins except Trp-Met-Asp-Phe in same experimental condition.

Secondly, we attempted to inhibit the in vitro growth of HEWL aggregates using sodium dodecylsulfate (SDS), cetyl trimethylammonium bromide (CTAB) and disulphide breaking agent, 1,4 dithiothreitol (DTT) in alkaline condition.

Halting the growth of diffusible protein oligomers in solution phase is needed to be developed for an effective therapeutic approach against pathogenic protein aggregates (May et al., 2006). We applied sensitive fluorescence techniques to track the growth HEWL in pH 12.2 alone and in presence of detergents (SDS, CTAB) and DTT. Time-resolved anisotropy decay reveals the faster segmental motion of dansyl conjugated HEWL in presence of SDS, CTAB and DTT at pH 12.2 after ~24 hrs of incubation, compared to HEWL alone in pH 12.2 (control). However, this rotational freedom was more pronounced in presence of DTT. High r

ss

value of control over the period of thirty ruled out any lag phase of HEWL in pH 12.2, whereas reduced r

ss

in presence of additives for the same time span confirmed their effectiveness in suppressing the growth of large HEWL aggregates. Presence of amyloid like structure and hydrophobic interaction was monitored by enhanced ThT and ANS fluorescence.

Substantial inhibitory action of CTAB and DTT against HEWL amyloid formation

was observed by reduced ThT fluorescence however, inhibitory action of SDS was

not possible to monitor due to background signal of ThT in presence of SDS. We also

monitored HEWL secondary structure in month old incubated sample at pH 12.2

alone and in presence of additives at room temperature employing circular dichroism

(CD). As enzymatic activity mirrors the native form of protein in any given sample,

we monitored the time dependent HEWL activity at pH 12.2 buffer alone and in

presence of SDS, CTAB and DTT. While revealing the importance of disulphide bond

during the process of HEWL aggregation at alkaline condition, free thiol groups were

found to increase for three days, and started decline after five days of incubation

suggested their potential involvement in stabilization of lysozyme aggregates.

I extended my work of inhibiting HEWL aggregation at pH 12.2 with another approach. In this part of work lysozyme was preincubated overnight at room temperature with its competitive inhibitor N,N′,N′′-Triacetylchitotriose (chitotriose) and N-Acetyl-D-glucosamine (NAG) before transferring to pH 12.2. After transferring to alkaline condition, reduced ANS and ThT fluorescence was observed at different time points in chitotriose incubated HEWL compared to sample at pH 12.2 without additive and in presence of NAG. Heterogeneity in HEWL aggregates was also observed in 12% SDS PAGE and we observed higher oligomers in 198 hrs old HEWL sample at pH 12.2 alone and in presence of NAG, but mostly monomeric species was observed at sample which was preincubated with chitotriose. Enzymatic activity of HEWL in same experimental conditions revealed ~75% activity in chitotriose incubated sample after 1500 minutes of incubation at alkaline condition which was nearly 24% in NAG incubated and almost zero in control (with no additives) for same time window.

While detecting HEWL amyloid in pH 12.2 in presence of additives, we came across a striking observation that enhanced ThT fluorescence was not an exclusive feature of amyloid. Negatively charged surfactant like SDS or cellular components also exhibits increased ThT fluorescence subsequent to their binding with positively charged dye. We monitored absorbance, fluorescence and steady-state anisotropy of ThT as a function of SDS concentration. Increments in all three parameters were observed up to 1.5 mM SDS which was saturated thereafter indicating their micellization. Surprisingly, absorption maximum of ThT was shifted in presence of SDS above its critical micellar concentration (CMC). When we tried to monitor the effect of positively charged CTAB or neutrally charged Triton X-100 and Tween 20 on the ThT absorption and emission spectra, we could not observed any changes over the wide concentration range of these surfactants. Thus comparing the absorbance and fluorescence of ThT with different surfactants, we observed that ThT selectively resides inside the anionic micelles like SDS instead of cationic and neutral micelles.

We also tracked the mammalian cells employing ThT fluorescence subsequent to 450 nm excitation.

Chapter 2

Experimental techniques

Brief introduction to molecular spectroscopy

Spectroscopy is a branch of science which identifies and measures interaction of electromagnetic radiation (ER) with matter. These interactions can be ascribed by absorption, emission or scattering. The transition of matter from a lower energy state to a higher energy states is the basis of absorption spectroscopy. The molecular transition strongly depends upon the frequency of the radiation absorbed. For instance, X-ray radiation which oscillates at a high frequency is energetic enough to excite core electrons, whereas lower frequency ultraviolet and visible radiation only excites valence electrons. Lowest energy transitions occur in the vibrational and rotational levels of molecules and nuclei and are induced by infrared and radio waves.

Emission spectroscopy, on the other hand, deals with the descent of an atoms or molecules from a higher energy state to a lower one. This transition is usually accompanied by the emission of radiant energy which can be measured by a variety of spectroscopic techniques specific to the frequency of the radiation. The various processes associated with the absorption and emission of radiation can be illustrated by Jablonski diagram (Figure 2.1).

Following the absorption of light, electrons are excited from the lowest energy level S0 to higher energy levels either S1 or S2. The energy difference between the two states is equal to the energy of the incident photon. The electronic transition occurs very fast with the time scale of 10^-15 seconds which is much faster than nuclear reorganization. Therefore, the higher energy states are achieved without any apparent change in the position of nuclei. This hypothesis is known as Franck-Condon principle. After absorption, a molecule returns to the ground state by radiative processes like fluorescence and phosphorescence and non-radiative processes such as vibrational relaxation, internal conversion, intersystem crossing, fluorescence quenching and other deactivation processes.

Figure 2.1 Jablonski diagram