doi:10.1016/j.bios.2005.06.010
Copyright © 2005 Published by Elsevier B.V.
Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology
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Torben Snabea, Gustav Andreas Røderb, Maria Teresa Neves-Petersena, Søren Buusb and Steffen Bjørn Petersena, 
, 
aAalborg University, Institute of Physics and Nanotechnology, Biostructure and Protein Engineering, Skjernvej 4C, DK-9220 Aalborg East, Denmark
bUniversity of Copenhagen, Department of Medical Microbiology and Immunology (IMMI), Panum, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
Received 26 April 2005;
revised 3 June 2005;
accepted 24 June 2005.
Available online 1 September 2005.
Abstract
Controlled and oriented immobilisation of proteins for biosensor purposes is of extreme interest since this provides more efficient sensors with a larger density of active binding sites per area compared to sensors produced by conventional immobilisation. In this paper oriented coupling of a major histocompatibility complex (MHC class I) to a sensor surface is presented. The coupling was performed using light assisted immobilisation—a novel immobilisation technology which allows specific opening of particular disulphide bridges in proteins which then is used for covalent bonding to thiol-derivatised surfaces via a new disulphide bond. Light assisted immobilisation specifically targets the disulphide bridge in the MHC-I molecule α3-domain which ensures oriented linking of the complex with the peptide binding site exposed away from the sensor surface. Structural analysis reveals that a similar procedure can be used for covalent immobilisation of MHC class II complexes. The results open for the development of efficient T cell sensors, sensors for recognition of peptides of pathogenic origin, as well as other applications that may benefit from oriented immobilisation of MHC proteins.
Keywords: MHC; Light assisted immobilisation; Oriented coupling; Biosensor
Fig. 1. The general principle of MHC-presentation of pathogenic peptides (antigen fragment) to the T cell via a T cell receptor. The CD4 or CD8 are T cell associated surface proteins that ensures tight binding to the target cell/the antigen presenting cell.
Fig. 2. MHC class I and class II sketched with domain notifications, disulphide bonds, and their association with the cell plasma membrane.
Fig. 3. The principle of light assisted immobilisation of a protein molecule sketched with tryptophan (Trp) near a disulphide bridge (cysteine sulphur atoms highlighted as large spheres). The surface can be gold or – as illustrated – a thiol-derivatised surface that can participate in the formation of a new disulphide bond between the surface and the protein.
Fig. 4. MCH class I protein (grey backbone) with microglobulin (black backbone) illustrated with the three disulphide bridges (comprised by cysteines in dark grey). Tryptophans are highlighted in light grey, and the bound peptide in black. One disulphide bridge is located in the microglobulin domain, one near the peptide binding site, and one in the membrane associating domain. The arrowed disulphide bridge (Cys203–Cys259) in the membrane binding domain is very close to Trp217 (4–5 Å), Trp204 (6–7 Å), and Trp244 (8–9 Å), and thereby is this disulphide bridge extremely disposed to reduction (and thus to the creation of free thiols which can couple to a thiol coated surface). Regarding the other disulphide bridges these are less disposed to light assisted reduction since they have only one tryptophan within 10 Å (see also Table 1). The model was created in WebLab Wiever Lite ver 4.0 (Molecular Simulation Inc.) based on PDB entry 1a1m.
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Fig. 5. MCH class II represented with the α-chain (grey backbone) and the β-chain (black backbone), the three disulphide bridges in dark grey (comprised by cysteines) and tryptophans in light grey. A bound peptide (black) signifies the binding site. The disulphide bridge in the membrane binding domains of the α-chain (Cys107–Cys163) and the β-chain (Cys117–Cys173) are both very close to two tryptophan residues each: Cys107–Cys163 is close to Trp121 (4–5 Å) and Trp178 (8–9 Å) and Cys117–Cys173 is close to Trp121 (4–5 Å) and Trp188 (10–11 Å). Thereby are these disulphide bridges extremely disposed to reduction (and to the creation of free thiols which can couple to a thiol coated surface). Regarding the disulphide bridges near the peptide binding site (Cys15–Cys79), the closest tryptophan is more than 13 Å away meaning that its disposition to reduction is very limited compared to the two membrane-associated disulphides. The membrane associated disulphide bridges are the most disposed heir disposition to significant reduction is limited (see also Table 1). The model was created in WebLab Wiever Lite ver 4.0 (Molecular Simulation Inc.) based on PDB entry 1fv1.
Fig. 6. (A) UV-assisted immobilisation of MHC complex on SH-activated quartz surfaces, analysed by TIRF. “Background immobilisation” was analysed by exposing the surface to protein in darkness. Fluorescence emission intensity at 350 nm was used as a measure of bound MHC complex. (B) Folding analysis of the immobilised MHC complex, analysed by TIRF. A MHC complex specific antibody conjugate (W6/32 labelled with Fluorescein) was used. Fluorescence emission intensity at 525 nm was used as a measure of bound antibody.
Fig. 7. The relative level of bound W6/32 mAb conjugate per MHC molecule (based on fluorescence data presented in Fig. 6).
Table 1.
Distances from disulphide bridges to their closest tryptophans based on crystal structures extracted from the Brookhaven Protein Data Bank
The domain notifications refer to location of the respective disulphide bridges (according to Fig. 4 and Fig. 5).
Corresponding author. Tel.: +45 96358469; fax: +45 96359129.
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