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Protein-Surfaces

Probing Protein dynamics on surfaces:

 
 
 

This research was initially funded (2003-08) by Science Foundation Ireland as part of a Principal Investigator award (02/IN.1/M231) and in part continued under the National Biophotonics Imaging Platform up to 2011 with Dr. Denisio Togashi being the lead researcher.

In 2012 an IRCSET funded PhD student (P. Zarski) continued with this research steam, developing Total Internal Reflection Microscopy (TIRFM) based methods for looking at protein-surface interactions.

In 2015 Camila van Zanten joined (funded by CAPES) and she is used advanced microscopy (FCS) to look at protein-nanoparticle interactions using FCS. 

Protein-Surface Interactions:

When a complex biological system (a body, a tissue type, living cell, etc.) interacts with materials (e.g. engineered materials such as implant devices) it does so chemically and that process can be difficult to observe on a molecular level.  The key interaction is that between the free proteins and the surface, and it is of major concern in a wide spectrum of fields such as biomaterials, nanotechnology, medicine, and biotechnological manufacturing processes.  There is a need not only to measure the amount and rate of protein adsorption, but also the changes in protein structure.  Whether protein adsorption is desirable or not, many fundamental features of protein-surface interactions still remain unknown, in particular information about protein structural changes during the adsorption process in complex environments. 

This prompts questions about protein folding/unfolding, denaturation, orientation, stability, conformation and activity when coupled to surfaces.  In many cases, these unknowns result from the fact that the tools are not readily available.  Exploiting this gap in the underlying principles is required in order to control or engineer the protein-surface interaction.  In this aspect, original ultra-sensitive approaches of detection, observation, and quantitative analysis must be developed and applied to provide meaningful, quantitative, and structural information.  Furthermore, these approaches and models must be robust enough to allow for the heterogeneous nature of proteins and surfaces, and also take into special consideration the real-time kinetics and thermodynamics of conformational changes that occur as a protein encounters a surface. Understanding the underlying, fundamental processes that govern the interaction of biological systems with surfaces is key to the development of biocompatible medical devices which lead to successful implantable devices.  This in turn translates to improved healthcare, minimally invasive surgery, healthier and prolonged lives.

In  the NBL we have been investigating the use of fluorescence based methods for characterising and quantifying protein-surface interactions.

Key Ref:  On the Adsorption of Proteins on Solid Surfaces, a Common but Very  Complicated Phenomenon. K.Nakanishi, T.Sakiyama, and K. Imamura J. Biosc. Bioeng. 91 (3) 233-244 (2001).

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Research:

Our research is focused on developing quantitative fluorescence based methods to measure and analyze the interaction of proteins with different surfaces in real time.  Some of the projects which we are undertaking (or have completed) include:

  • Protein-Nanoparticle Interactions:  Developing fluorescence based methods to study the interaction of nano-particles with proteins.  This was part of a CAPES and a Hardiman funded PhD scholarship awards.
  • Protein-Surface Interactions:  Quantifying the amount of protein adsorbing on surfaces & understanding structural changes using advanced fluorescence microscopy (Total Internal Reflection Fluorescence).
  • Protein-Protein interactions in living cells:  As part of the Systems Biology Ireland Initiative we collaborated with Dr. H.-P. Nasheuer to measure protein-protein interactions in live cells using Fluorescence Correlation Spectroscopy (FCS).  This is now concluded.

Current researchers:


Past senior researchers:

Dr. Denisio Togashi (2004-2011):  protein-surface interactions.
Domhnall O'Shaughnessy (PhD student, protein-nanoparticle interactions).
Przemyslaw Zarski (PhD student, 2012-18):  protein-surface interactions measured using TIRF microscopy.
Camila Van Zanten (PhD student, 2015-20):  protein-nanoparticle interactions measured using FCS microscopy.

Other Contributing researchers & students:

2009: Loretta Breslin  and Neil Murphy (MSc project students), Edel Houghton (UREKA summer student)
2008: Amandine Calvet (project student), Valerie Murphy (4Y student).
2007: Muireann O'Loughlin (4Y student), Noemie Marguerite (French Undergraduate), Emmanuelle Bays (Swiss, IAESTE trainee.)
2006: Deirdre McMahon (MSc project student).
2005: Margaret Collins (MSc project student).

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Publications:

  1. Estimating Poly(N-isopropylacrylamide) size in solution below the LCST using Fluorescence Correlation Spectroscopy with non-covalent bound fluorophores.  C. van Zanten and A.G. Ryder. Polymer Testing,  137, 108500, (2024). DOI:   10.1016/j.polymertesting.2024.108500 [Open access]
  2. Effects of viscosity and refractive index on the emission and diffusion properties of Alexa Fluor 405 using fluorescence correlation and lifetime spectroscopies., C. van Zanten, D. Melnikau, and A.G. Ryder.  Journal of Fluorescence, 31(3), 835-845, (2021). DOI: 10.1007/s10895-021-02719-y 
  3. Super Stable Fluorescein Isothiocyanate Isomer I Monolayer for Total Internal Reflection Fluorescence Microscopy.   P. Zarski and A.G. Ryder.  Langmuir, 34(37), 10913-10923, (2018).  DOI: 10.1021/acs.langmuir.8b02509
  4. Cell cycle-dependent mobility of Cdc45 in living cells determined by fluorescence correlation spectroscopy.  R. Broderick, S. Ramadurai, K. Toth, D. Togashi, A. G. Ryder, J. Langwoski, and H.P. Nasheuer.  PloS One,  7(4): e35537, (2012).  DOI: 10.1371/journal.pone.0035537. 
  5. Assessing protein-surface interactions with a series of multi-labeled BSA using Fluorescence Lifetime Microscopy and Förster Energy Resonance Transfer.  D.M. Togashi and A.G. Ryder, Biophysical Chemistry, 152, 55-64, (2010).  DOI:  10.1016/j.bpc.2010.07.006
  6. Monitoring Local unfolding of Bovine Serum Albumin during denaturation using steady-state and time-resolved fluorescence spectroscopy.  D.M. Togashi, A.G. Ryder, and D. O'Shaughnessy, Journal of Fluorescence, 20(2), 441-452, (2010). DOI: 10.1007/s10895-009-0566-8
  7. Quantifying Adsorbed Protein on Surfaces using Confocal Fluorescence Microscopy. D.M. Togashi, A. G. Ryder, and G. Heiss,  Colloids and Surfaces B: Biointerfaces. 72(2), 219-229, (2009).
    DOI: 10.1016/j.colsurfb.2009.04.007
  8. Investigating trypthopan quenching of fluorescein fluorescence under protolytic equilibrium.  D.M. Togashi, B. Szczupak, A.G. Ryder, A. Calvet, and M. OLoughlin,  Journal of Physical Chemistry A, 113(12), 2757-2767, (2009).   Online at ACS: http://pubs.acs.org/doi/full/10.1021/jp808121y .
  9. Trigger factor from the psychrophilic bacterium P. Frigidicola is a monomeric chaperone.  S. Robin, D.M. Togashi, A.G. Ryder, and J.G. Wall, Journal of Bacteriology, 191(4), 1162-1168, (2009).
    Online here. DOI: 10.1128/JB.01137-08 
  10. A fluorescence analysis of ANS bound to bovine serum albumin: binding properties revisited by using energy transfer.  D.M. Togashi and A.G. Ryder.  Journal of Fluorescence, 18(2), 519-526, (2008).  
    Online here. DOI: 10.1007/s10895-007-0294-x.
  11. Fluorescence Lifetime Imaging study of a thin protein layer on solid surfaces. D.M. Togashi and A.G. Ryder.   Experimental & Molecular Pathology, 82(2). 135-141, (2007).
    DOI: 10.1016/j.yexmp.2007.01.005
  12. Mobility and distribution of replication protein A in living cells at single molecule level.  C. Braet, H. Stephan, I. Dobbie, D. Togashi, A.G. Ryder, Z. Foldes-Papp, N. Lowndes, and H.P. Nasheuer.  Experimental & Molecular Pathology, 82(2). 156-162, (2007).  DOI: 10.1016/j.yexmp.2006.12.008
  13. Time-resolved fluorescence studies on bovine serum albumin denaturation process.  D.M. Togashi and A.G. Ryder,  Journal of Fluorescence, 16(2), 153-160, (2006). 
    DOI:  10.1007/s10895-005-0029-9

 

Conference Presentations: 

  1. Using Fluorescence Correlation Spectroscopy (FCS) to measure protein stabilization by PNIPAm nanoparticles under mechanical stress.  C. van Zanten, D. Melnikau, and A.G. Ryder, Joint 12th EBSA, 10th ICBP-IUPAP Biophysics Congress, Madrid, 20-24 July, 2019. [Poster]
  2. Investigating the interaction of Alexa Fluor 405 and Atto 390 with PNIPAm using fluorescence correlation spectroscopy (FCS). C. Van Zanten,* D. Melnikau, A.G. Ryder.  15th Conference on Methods and Applications of Fluorescence: Spectroscopy, Imaging and Probes, MAF15, Bruges, Belgium, 10-13 Sept. 2017.
  3. Fluorescence Correlation Spectroscopy (FCS) studies of PNIPAm and Atto 390 systems in aqueous solutions.  C. van Zanten,* D. Melnikau, and A.G. Ryder, NUI Galway/UL Alliance Research Day, 19th April, 2017.
  4. Monitoring adsorption of fluorescein iso-thiocyanate isomer I (FITC) on modified glass surface by total internal reflection fluorescent (TIRF) microscopy, P.M. Zarski,* A.G. Ryder, 14th Conference on Methods and Applications of Fluorescence: Spectroscopy, Imaging and Probes, Würzburg, Germany, 13–16 Sept., 2015.
  5. Using Total Internal Reflection Fluorescence Microscopy (TIRFM) for studying protein-surface interactions, 13th Conference on Methods and Applications of Fluorescence: Spectroscopy, Imaging and Probes, Genoa, Italy, 8 - 11 Sept., 2013.
  6. Study of Protein Deposition on Co-Polymers with different wetabilites by Confocal Fluorescence Microscopy, L. Breslin, D.M. Togashi, and A.G. Ryder, 42nd IUPAC Congress, Glasgow, 2-7 Aug. 2009.
  7. Probing the Structural Changes of Serum Albumin Protein in Thin Adsorbed Layers on Hydrophilic/Hydrophobic Surfaces using a FLIM-FRET approach.  D.M. Togashi and A.G. Ryder.  International Conference on Trends in Bioanalytical Sciences and Biosensors ( ICTBSB-2009), Dublin, 26-27 January 2009.
  8. Investigating Intramolecular Fluorescence Quenching in Serum Albumin Protein.  M. O’Loughlin, D.M. Togashi, A.G. Ryder Europtrode IX, Dublin, March 30 - April 2, 2008.
  9. Confocal Fluorescence lifetime imaging (FLIM): a tool for analysis of structure changes of protein adsorbed onto solid surfaces. D.M. Togashi and A.G. Ryder.   10th Conference on Methods and Applications of Fluorescence: Spectroscopy, Imaging and Probes, Salzburg , Austria , 9-12 Sept., 2007.
  10. Binding Properties Revisited: a fluorescence analysis of ANS bound to bovine serum albumin.  D.M. Togashi and A.G. Ryder.  10th Conference on Methods and Applications of Fluorescence: Spectroscopy, Imaging and Probes, Salzburg , Austria , 9-12 Sept., 2007. Poster available here.
  11. Fluorescence study of Bovine Serum Albumin and Ti and Sn Oxide Nanoparticles Interactions.  D.M. Togashi, A.G. Ryder, D. Mc Mahon, P. Dunne, and J. McManus.  European Conference on Biomedical Optics, Munich, Germany, 17-22 June, 2007. 
  12. Evaluating the Structure Changes of Albumin Adsorbed onto Solid Matrix with Different Wettabilities by Fluorescence Lifetime Imaging (FLIM).  D. Togashi and A. Ryder,  7th International ELMI meeting, York, England,  17-20 April, 2007.
  13. FCS and Fluorescent proteins: measuring diffusion rates, concentrations and protein-protein interactions.  I. Dobbie, H. Stephan, H.-P. Nasheuer, D. Togashi, A. Ryder, and N. Lowndes.  7th International ELMI meeting, York, England,  17-20 April, 2007.
  14. Application of fluorescence lifetime imaging on adsorption of bovine serum albumin on solid surfaces.  D.M. Togashi and A.G. Ryder.  Microscopical Society of Ireland's 30th Annual Symposium, NUI-Galway, 30 Aug.-1 Sept., 2006.
  15. Real-time measurement of adsorption of bovine serum albumin on silica glass by confocal fluorescence microscopy. D.M. Togashi and A.G. Ryder.  6th International ELMI meeting, Ofir , Portugal , 30 May-2 June, 2006. 

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Methods for Studying Protein Surface Interactions:

This will be updated in the coming months.  FLIM.  TIRF. 

TOC graphic for FITC monolayer paper.Total internal reflection fluorescence microscopy (TIRFM) is an important method in surface science and for the analysis of surface bound macromolecules. Here we developed and explored the use of a novel fluorescein isothiocyanate isomer I (FITC) adsorbed monolayer for alignment and validation of TIRFM measurements and configurations. Aqueous solutions of FITC exist as several different protolytic forms (di-anionic, anionic, neutral, and cationic) with each form having different emission characteristics. However, the emission behavior of FITC adsorbed on hydrophilic, hydrophobic, and unmodified glass surfaces at different pH was unknown. TIRFM imaging and spectroscopy were used to study FITC and FITC labelled Bovine Serum Albumin (BSA-FITC) monolayers generated on three different glass surfaces. Monolayer emission intensity, spectra, and the photobleaching profiles were all dependent on pH and the surface properties of the glass. Very strangely however, at pH 5.0 on hydrophobic surfaces the FITC monolayers produced were both bright and apparently un-bleachable over ~20 minutes of imaging (60 sec. total exposure). During monolayer formation at pH 5.0 we saw clear evidence for concentration-based quenching, indicating high surface coverage. When the monolayer had been rinsed with buffer to remove unbound FITC we observed an increase in emission intensity during illumination indicative of some form of photoactivated species being present. Eventually the fluorescence emission stabilized and remained constant for extended periods of time with no evidence of photobleaching. We hypothesize that during the adsorption process (a hydrophobic–hydrophobic interaction) there was conversion to the fluorescent quinoid form of FITC [1]. In contrast, at pH 7.4 and 9.6 on hydrophobic surfaces, FITC monolayers had well-defined, fast photobleaching kinetics (decay to ~50% intensity in 5-10 seconds). The equivalent BSA-FITC monolayers were somewhat brighter, with similar photobleaching kinetics. While the precise mechanism for this unusual behavior is still unknown, all these low-cost monolayers were easily prepared, were reproducible, and can serve as convenient test samples for TIRFM alignment, calibration, and validation prior to undertaking measurements with more sensitive biogenic or biological specimens.

  1. Stable FITC and protein monolayers for Total Internal Reflection Fluorescence Microscopy, and the super-stable exception:  an un-bleachable monolayer?   P. Zarski and A.G. Ryder.  Langmuir, 34(37), 10913-10923, (2018).  DOI: 10.1021/acs.langmuir.8b0250
 

 
We also have a range of other research projects where the equipment could be used for protein-surface interaction measurements, a more detailed list of equipment is given on the Instrumentation Pages.

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Information Links:

These are some of the sites that we regularly use. I hope to add more links and details in the near future. If there are problems with any of the links let me know.

Technical Journals & Societies:

Surface Research & courses:

Polymer-protein surfaces, Switzerland.
Proteins-Polymers-Interfaces Group, at the Department of Bioengineering at the University of Utah.

Information Resources:

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Equipment Links:

This is a selection of useful web-sites from a range of manufacturers who produce the equipment we use.

Olympus TIRF.
Ocean Optics (for fiber coupled spectrometers)
Ibidi (for disposables and chambers)

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