Fluorescence emission of Immunoglobulin G is determined by the presence of 11 Trp and 28 Tyr residues. 1 Trp and 11 Tyr are involved into emission of Hu-man Albumin (2 Trp for BSA). Trp and Tyr residues are distributed in different protein local environment. Some of the residues are exposed to solvent, others are buried. It is known that Trp can present rotamers [102], resulting in different ori-entation of indole to peptide bond which will then influence the dynamics of relax-ation. Different environments of Trp residues in Immunoglobulin G and Albumin lead to a shift in fluorescence emission. The emission maximum depends on local Trp position in protein polypeptide chain, i.e. its proximity to solvent molecules, quenching, substrate binding, folding/unfolding, etc. The mechanism of this
ef-0 5 10 15
Figure 5.17:Discrimination of rabbit IgG against BSA under single channel optimization. Excitation UV - 295 nm. a) Rab-bit IgG; b) BSA. Blue: fluorescence depletion traces obtained with unshaped UV pulse. Red: fluorescence depletion traces measured with shaped UV pulse. Ex-citation wavelength is set at 295 nm.
fect is theoretically investigated by Vivian and Callis [104] using hybrid quantum mechanical/molecular dynamics technique. They observe that when indole
inter--600 -400 -200 0 200 400 time [fs]
Figure 5.18:X-FROG trace for retrieved optimal pulse obtained dur-ing the close-loop optimization for discrimination of rabbit IgG and BSA.
acts with solvent and surrounding protein residues, it induces rearrangement of the Trp ring electron density that subsequently affects the transition energy and thus fluorescence emission.
Our results demonstrate the sensitivity of the depletion peak to its local protein environment: the amplitude as well as relaxation time of the peak varies for dif-ferent excitation wavelength and studied protein. The measurements performed with time-resolved pump-probe spectroscopy pumped at 270 nm excitation indi-cate distinct excited-state dynamics of human IgG and HSA resulting in different relaxation times:τ1=0.14±0.13for IgG andτ1'0.99±0.41for HSA. The rel-ative amplitudes correspondent to these decays areA1=0.19±0.29 andA2=0.92
±0.01 for human IgG, respectively for HSAA1=0.10±0.02 andA2=0.85±0.01 . On the other hand, at 295 nm excitation protein dynamics are slower according to our measurements with UV pump pulse close to FT-limited, exhibitingτ1=4.50
±2.34 ps with the relative amplitudesA1=0.08±0.02 and A2=0.87±0.01 for rabbit IgG, for BSAτ1=5.61±2.23 ps with relative amplitudesA1=0.12±0.02 andA2=0.82±0.01 . At this excitation we observe increase in amplitude of a dip appeared at short time delays near 1 ps.
In an attempt to interpret the observed behaviour, two possible mechanism can be proposed. The first is the effect of the solvent and surrounding protein residues.
The difference in the relaxation times for IgG and HSA, as well as for IgG rabbit and BSA irradiated at 295 nm, may be the reflection of different local electro-static environment of indole chain of Trp. A similar effect is reported by A.Rondi during the investigation of Trp and Ala-Trp dynamics in different solvents [134].
In Dioxane at 270 nm excitation, for instance, a similar short relaxation time of τ ∼0.35±0.01ps for Ala-Trp andτ ∼0.10±0.32ps for Trp, is observed which is evidence of a correlation between the dielectric constant of the solvent, relax-ation time and the Stokes shift. The solvrelax-ation dynamics of Trp in serum proteins [163,156], in particular HSA [156], was investigated and reported in the series of articles by by Zhong and co-workers. They observe that for HSA solvent relaxation is much slower than for Trp. For different conformation rotamers relaxation time is 1-4 ps and 20-30 ps [156]. However, they did not observe the faster component which is predicted by MD simulations. Another study of water-protein interac-tions of proteins performed by group of A. Zewail shows that hydration dynamics of proteins is represented by exponential decays with time scales of 0.2-0.8, 1.4-6.1, and 10-61 ps [164]. Notably, the longer component of 10-61 ps is missing for Trp, which is related to collective water network rearrangement coupled to protein fluctuation dynamics [164,165,166,167,168].
The energy transfer is another mechanism that may be attributed to the different relaxation times and the significant decrease coherent peak amplitude. Indeed, under irradiation of 270 nm both aromatic amino-acids Trp and Tyr are excited.
At this wavelength of excitation, we should consider an energy transfer between Tyr and Trp residues, where Tyr fluoresce at a short spectral region of 308 nm.
As demonstrated previously by A.Rondi [10], these amino-acids exhibit different excited state dynamics when probed by IR pulses: Trp has very sharp coherent peak attributed to the opening of a Franck-Condon window, towards higher ionizing and dissociative states, which, in contrast, is lacking for Tyr. Orientation of the indole relative to peptide bond, as well as an energy transfer between Trp and Tyr residues can be an explanation for the absence of strong dip characteristic for Trp-containing dipeptides and Trp alone.
A comparison with results obtained under 295 nm excitation reveal a possible explanation for this observation. In this case only Trp residues contribute to the flu-orescence, and tyrosine emission is negligible. Consequently, we observe a small coherent peak at short time delays which is detected for both serum proteins. The
comparison in dynamics of different IgGs under excitation of 270 nm and 295 nm can be explored in Figure 5.19. Both depletion traces are plotted on the same fig-ure. Note, Figure 5.19 depicts human IgG under excitation of 270 nm and rabbit IgG at 295 nm excitation. We confirm that dynamics of rabbit IgG are similar to human IgG at both wavelengths of excitation. Therefore, the dynamics are still much faster for both proteins than for Trp and Trp-containing dipeptides alone.
-4 -2 0 2 4 6
0.0 0.2 0.4 0.6 0.8 1.0
Normalized depletion signal, a.u.
Time-delay, ps
Figure 5.19:Fluorescence depletions of IgG under the excitation with FT-limited pulse at 295 nm (for rabbit IgG) and 270 nm (for human IgG).
In spite of similarities in depletion signal at short delays between the pump and probe displayed by IgG and albumin, in both experiments (pump at 270 nm and at 295 nm ) optimal control strategy allows us to increase discrimination power. In the first case, where excitation is at 270 nm, the optimal pulses are employed for the further identification of immunoglobulin in human serum (reported in the following chapter). The fluorescence depletion of IgG decreases upon the excitation with an optimal pulse, while the increase in the signal is observed for HSA. This reflects the wave-packet motion toward the region of the potential energy surface where the
transition dipole moment is high for HSA and lower IgG, so that we observe the lower depletion signal. For IgG coupling of the shaped UV pulse with vibrational modes inS1state probably is faster intra- and intermolecular energy redistribution, hence coherently excited vibrational modes are rapidly depopulated.
A pulse shaper generated a multi-pulse sequence for all the optimizations. The presence of a multi-pulse structure responsible for IgG and HSA discrimination may indicate a role played by excited-state vibrational resonances. We examine the FROG spectrogram with Fourier Transform analysis, and obtained the follow-ing frequencies extracted from the correspondfollow-ing pulse train:122cm−1,140cm−1, 98cm−1,95cm−1for IgG human and HSA. A multi-pulse structure is also obtained for IgG rabbit and BSA irradiated at 295 nm. The frequencies obtained with an X-FROG measurement may indicate vibrational modes on the excited-state surface targeted by optimally shaped pulse. However a direct comparison with such ex-cited states modes is difficult, because it would need a dedicated experiment.In this regard, measurements with THz or far IR spectroscopy are desirable to correlate vibrational spectra with obtained frequencies of the optimal pulse, as it was made in the [75].
Transient absorption measurements and also 2D UV spectroscopy could give contribution to reveal the underlying mechanism leading to protein discrimination upon the excitation of optimally shaped pulse. These techniques are very powerful to monitor protein dynamics and local structural changes. Therefore they can be employed to analyse processes like folding, energy and electron transfer between different residues that lead to Trp fluorescence quenching. Experiments with bac-teriorodopsin performed by group of Prof. M. Chergui using transient absorption setup demonstrated the sensitivity of Trp absorption band to ultrafast laser field [169]. They could observe progressive dipole moment changes within protein in-duced by ultrafast laser field by monitoring the absorption changes of Trp residues.
At this step we suggest possible plausible pathways affected by optimal pulse to-wards serum protein discrimination. The structure of Immunoglobulin is composed in such a way that Trp residue is in close proximity to disulphide bridges, which is considered to be a strong quencher [170]. They have a distance no longer than approximately 4.5 A [171]. It is also known that protein UV irradiation results in formation of electron or H-atom ejection from Trp and Tyr. This can contribute to
the cleavage of the cystine residues formatting disulphide bridges. Several mech-anisms have been proposed in this respect. The mechmech-anisms, responsible for de-struction of disulphide bridges, relies on the electron transfer from the excited state Trp to the disulphide bridges. The second mechanism is: 1) UV irradiation of Trp induces excited state dipole moment which subsequently induces dipole moment in the adjacent disulphide bond [172]; 2) energy can be transferred from excited Trp to the disulphide bond, which then can induce vibrational modes leading to disruption of the disulphide bond. It has been reported that the probability of disul-phide bridge disruption is independent of light intensity and protein concentration [172]. In fact, this property of photochemical reaction between Trp and disulphide bridges lead to a potential biosensing application to immobilize protein on solid surface for detection of the antigen [173,174].
5.5 Conclusions
We have presented the extension of optimal control for complex molecular sys-tems, serum proteins: human IgG and HAS, human IgG and human IgM, rabbit IgG and BSA. We investigated proteins by means of time-resolved fluorescence and steady-state spectroscopies. Various dynamics was observed for studied anti-bodies and serum albumins, when they are irradiated at two different wavelength, 270 nm and 295 nm. Difference in the relaxation time constants of the sample was attributed by possible energy transfer between Tyr and Trp residues.
Successful discrimination was achieved for the molecular pairs of human IgG and HSA, and rabbit IgG and BSA, as a results of the single channel optimiza-tion. For the couple of proteins, human IgG and HSA, the observed effect of the fluorescence difference between the optimized pulse shapes, was of the order of 8.5σ. For the couple of antibodies, human IgG and human IgM, the observed ef-fect was much lower of the order 2.8σ(if we compare depletion ratios of the results obtained with optimally shaped pulse and with FT-limited). We attempted to inter-pret the cause, leading to the discrimination. However, the underlying mechanism remains undefined. More detail study is needed to fully understand variance in protein dynamics induced by optimally shaped pulses. Spectroscopies, for exam-ple NMR, Raman and FTIR are good candidates, which could be included in future studies. Combination of one of these techniques with computer simulations might be beneficial in predicting of successful outcome of the GA optimization.
In the next Chapter, we will demonstrate the application of optimally shaped pulse towards protein identification in the mixture, which is very demanding for the health diagnostics.
ODD application for protein identification
6.1 Introduction
Human serum contains a large number of proteins including antibodies, serum albumin family (albumin), globulin family (hemoglobin, myoglobin), hormones, lipoproteins, and other compounds (see Chapter 1.1). So far various biomolecular screening techniques have been developed to study protein function and protein identification in human serum for the diagnosis of diseases. The most common bio immunoassays rely on antibody-antigen interaction, and some of the diagnostics methods are reported in the Chapter 1.3. Strong specificity of such interactions has led to the development of a variety of bioassays able to detect the presence of either antibody or antigen. Many of them use some specific labelling agents, which means adding additional secondary antibodies or fluorescent, chemical and radioactive tags. However the development of label-free biosensors, which eliminate the use of any additional label, opens up new horizons in this field. Among their advantages are the fast development time of the assay, high accuracy, real-time readout and less interference with the sample.
ODD technique is a good candidate for a diagnostic purpose like label-free sens-ing. Designing an optical database containing optimal UV pulses with the spectral phases specific for certain proteins will greatly aid protein identification and the study of their interactions with other biomolecules.
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