TIRF and its application to protein adsorption : electrostatics and orientation

The aim of the study in this thesis was to develop a method for determining the orientation of adsorbed protein molecules and to study the influence of the electrical potential of the interface on the interfacial properties of proteins, including their orientation.In the adsorption of proteins on solid surfaces many factors play a role. The most important are electrostatic and hydrophobic interactions between the protein molecules and the sorbent surface, and structural rearrangements in the protein molecules. From earlier studies it was concluded that proteins with regard to their adsorption behaviour can be roughly divided into "hard" and "soft" proteins. For the "hard" proteins structural changes upon adsorption are negligible and these proteins do not adsorb on hydrophilic surfaces unless there is electrostatic attraction. "Soft" proteins have a tendency to unfold partially upon adsorption and they adsorb on all kind of surfaces, irrespective of any electrostatic repulsion, partly caused by a gain of conformational entropy during adsorption.The orientation of adsorbed protein molecules plays an important role in the effectivity and the development of immunoassays and diagnostic tests. One can imagine that if the orientation of an antibody (or enzyme) is not the right one, no recognition of the antigen (or substrate) occurs. Therefore, either much research is done to develop methods to adsorb antibodies and enzymes in the proper orientation or in ways to steer the adsorption process. In the development of biosensors knowledge of and insight into the adsorption process and the orientation of the adsorbed proteins molecules (used as the selector molecules) on inorganic materials also play essential roles.In this study two optical techniques have been used: Total Internal Reflection Fluorescence (TIRF) and reflectometry. With both techniques it is possible to monitor quantitatively and qualitatively the adsorption process of proteins in situ.TIRF has been used over the past 20 years for the measurement of protein adsorption kinetics and adsorbed amounts, and to study the exchange of proteins between sorbent surface and solution. A relatively new research topic for which TIRF is used is to obtain information on the orientation of adsorbed (protein) molecules. The principle of TIRF is as follows: A light beam is totally reflected at an interface between two media I and 2 with the refractive index of medium I higher than that of medium 2, and the angle of incidence exceeding its critical angle value. Due to interference between the incident and the reflected light beam an evanescent wave penetrates into medium 2. The amplitude of this electromagnetic wave decays exponentially with distance normal to the interface. The penetration depth of this wave depends on the wavelength of the light used, and the refractive indices of the media. For visible light striking a quartz/water interface it is in the order of 100 nm. If medium 2 consists of a solution with fluorescent molecules, the evanescent wave will excite the molecules that are close enough to the interface; the emitted fluorescence is detected. In the case of adsorption and not too high bulk concentrations, the fluorescence signal is almost completely stemming from molecules in the adsorption layer.With the optical technique reflectometry the adsorption of molecules on an (optically flat) solid surface can be monitored. A linearly polarized light beam is reflected from the adsorbing surface, and the reflected beam is split into its parallelly and perpendicularly polarized components. The intensity ratio between these two components is measured continuously. This ratio changes upon adsorption, and after calibration the adsorbed amount (mass/area) is obtained.In this work TIRF has been used for the determination of the orientation distribution of adsorbed molecules. Therefore, the existing theory had to be extended (chapter 2). Cytochrome c has been chosen as a model protein to test and illustrate the method, because of its welldocumented crystallographic structure and its well-characterized physicalchemical properties. Furthermore, cytochrome c has a chromophoric group which can be made fluorescent by removing the Fe-atom. The protein without the Fe-atom is called porphyrin cytochrome c. From various literature data it is inferred that the native cytochrome c molecule is rather structure-stable. Another interesting feature of the protein is its relatively large electric dipole moment (325 Debye at pH 7), which might offer a possibility to influence the orientation in the adsorbed state by variation of the surface charge.The method for determination of the orientation of adsorbed molecules is based on the principle that by changing the polarization of the incident light beam the direction of the electric field component of the evanescent wave is modified. As a result the interaction between the transition dipole moment of the adsorbed molecules and the evanescent wave alters, which in turn, gives rise to a change in the fluorescence intensity. To obtain order parameters from which the orientation distribution can be reconstructed, one has to measure not only the intensity but also the polarization of the fluorescence as a function of the polarization of the incident light beam. The theory has been elaborated especially for orientation measurements on porphyrins and cytochrome c. In the porphyrin ring two transition dipole moments are lying perpendicularly to one another. For this system it is possible to study the orientation distribution in one orientation angle from the restricted information: the angle 0 between the plane of the porphyrin ring and the interface. The orientation distribution in θcan be reconstructed using the Maximum Entropy Method. With regard to the mobility of the adsorbed molecules, which might interfere with the orientation measurements, it is shown that rotational mobility much faster than the fluorescence lifetime would result in the disappearance of the fluorescence polarization.Firstly, some experiments with a simple porphyrin (tetramethyl-pyridiniurn porphyrin, H ₂ TMPyP) have been performed (chapter 4). Prior to the orientation measurements, its adsorption behaviour was studied by reflectometry. For adsorption on silica from pure water, from 0.01 M phosphate buffer pH 7 and from 0.1 M KNO ₃ solution different adsorbed amounts have been found. From the TIRF orientation measurements satisfying results were obtained, although the reproducibility leaves still something to desire. The orientation distribution of adsorbed H ₂ TMPyP molecules on silica depends on the concentration of porphyrin in the solution from which adsorption takes place. At low concentration, the H ₂ TMPyP molecules are more or less randomly oriented, while at high concentrations a broad distribution around an angle of 46° between the porphyrin plane and surface was observed. The fact that the fluorescence is polarized and the results of measurements with different solution viscosities show that the mobility of the adsorbed porphyrin molecules is on a much larger time scale than the fluorescence life time (5 ns).To study the influence of the electrical potential (charge) of the sorbent surface on the adsorption behaviour of proteins, a semi-conducting indium tin oxide (ITO) surface was used. This material was deposited in a thin layer (120- 140 run) on glass or silicium plates. The ITO surfaces have been characterized by streaming potential measurements, scanning electron microscopy, atomic force microscopy and resistance measurements. The results have been described in chapter 3.The adsorption behaviour of various proteins (serum albumin, lysozyme, ribonuclease A, superoxide dismutase, myoglobin and α-lactalbumin) as a function of an externally imposed interfacial potential has been studied using reflectometry (chapter 5). The sorbent surface was again a semi-conducting ITO layer deposited on a silicium wafer. The results obtained at the equilibrium potential as a function of pH suggest that electrostatic interactions play a decisive role in the adsorption of structurestable proteins on hydrophilic surfaces. On the other hand, protein adsorption is found to be hardly affected by externally imposed interfacial potentials, irrespective of the structure-stability of the protein. The cause for the apparent contradiction in these results must be that in both experimental approaches, but in different ways, together with the electrostatic interactions other properties of the system are also varied. (For example, on changing the pH, the net charge of the protein molecules changes, but also their structure-stability; together with increasing surface potentials, the surface becomes more hydrophilic; and, in the case a constant potential is externally applied, the adsorbing protein molecules may largely adapt their properties.) Therefore it is difficult to assess the importance of the contribution of electrostatic interactions in the process of protein adsorption. Presumably, in the past protein adsorption as a function of the pH has been interpreted in a too simplified manner, overestimating the role of electrostatic interactions.In chapter 6 the adsorption behaviour of native and porphyrin cytochrome <em>c</em> was the subject of study. Special attention is given to the adsorbed amounts, the adsorption kinetics and the influence of externally applied potentials for both forms of cytochrome c and the orientation of adsorbed porphyrin cytochrome c molecules. It was shown that the adsorption behaviour of native cytochrome c resembles that of structurestable proteins such as lysozyme and ribonuclease. In many aspects porphyrin cytochrome c behaves the same as the native form. However the adsorbed amounts at pH 7 and 10 are much higher. The adsorbed amounts and adsorption kinetics of both forms of cytochrome c are found to be hardly affected by externally imposed potentials. With regard to the orientation measurements it was not possible to interpret the data in terms of orientation distribution functions because of the scatter in the results.This spread is mainly caused by the low signal to noise ratio. Improvement of this ratio is difficult because of photo-deterioration of the adsorbed protein molecules. However, the total fluorescence as a function of the polarization angle of the incident light beam points to orientation distributions which do not depend on the surface coverage and cannot be influenced by imposing an electrical potential on the sorbent surface.In summary, in this work it is shown that TIRF is a suitable technique to determine orientation distributions of adsorbed fluorescent molecules. Further-more, it was found that it is not (or hardly) possible to influence the adsorption behaviour of proteins, irrespective of their structural stability, by externally imposing an electrical potential to the sorbent surface.<strong>Perspectives</strong>In this work we have developed a method to obtain the orientation distributions of adsorbed chromophores by making use of the optical technique TIRF. The results obtained with a simple porphyrin show that the method works. So far, application of this method to adsorbed proteins is limited, since the protein should be structure-stable upon adsorption and carry a fluorescent group. It was not possible to reconstruct the orientation distribution of cytochrome c molecules because of the scatter in the order parameters obtained. In order to obtain the orientation distribution it is necessary to improve the signal to noise ratio of the fluorescence measurements. This could be done by measuring the fluorescence over a longer time. In the case of cytochrome c this fails because the adsorbed molecule appears to undergo structural rearrangements in the presence of light.For other structure-stable proteins, it might be possible to determine the orientation distribution in the adsorbed state. If the molecules do not have a fluorescent group, one might consider to label them; prerequisite is that the fluorescent label is fixed in the molecule with a known orientation to the rest of the molecule. However, a caveat is that introducing a fluorescent label might lead to structural changes within the molecule and hence influence the adsorption behaviour of the protein. Another possibility to obtain orientation distributions of adsorbed proteins with TIRF is to make use of the fluorescence of the aromatic amino acids tryptophan and tyrosine. These amino acids have a fixed place in the structure and their excitation wavelength is in the UV. This sets higher demands to the optical parts in the experimental set-up. Furthermore, the presence of more than one of these amino acids can cause energy transfer from one amino acid to the other. As a result it is not known where the fluorescence is stemming from and the information about the orientation is lost. Another disadvantage is that protein molecules might be damaged by the UV light.Meanwhile, the TIRF method for determining the orientation of adsorbed chromophores is already used in a study concerning the development of "organic" solar energy cells, conducted in the Department of Molecular Physics of the Wageningen Agricultural University. In this study porphyrin molecules are used as sensitizers to generate charge carriers in a semiconducting surface. The adsorption of porphyrins on this surface, especially their orientation, is a prominent factor determining the efficiency of the system. The more parallel the molecules lie on the surface, the higher the energy transfer is. The results obtained here with H ₂ TMPyP and presented in chapter 4 were promising enough to use the method for studying the orientation of several derivatives of H _{<BLINK>2</BLINK>} TMPyP.In our own department the method is now also used to investigate the order in Langmuir-Blodgett (LB) layers of phospholipids, which stand model for biological membranes. In the near future the structure and permeability of such phospholipid layers will be studied as a function of the electrical potential of the substrate. To that end, the LB layers will be deposited onto optically transparent conducting ITO films on quartz slides. With different fluorescent probes it is hoped that information on the rotational mobility and/or reorientation is obtained.Integration of TIRF with time-resolved fluorescence measurements can provide more detailed information on the structure and orientation of adsorbed molecules and on dynamic processes taking place on the time scale of fluorescence, e.g. rotation of the whole molecule or parts of it.In conclusion, the potentials of TIRF to study orientations are not exhausted.