The structure of not one integral membrane bound mammalian 7-TMD receptor has yet been crystallised nor the structure been determined. However, much has been inferred from the primary sequence of such receptors. A readily available example, rhodopsin, the vertebrate and invertebrate photoreceptor protein from retina, is being used as a model system to gain an understanding about how these proteins function. This project's aim has been to develop highly novel, state-of-the-art NMR methods to further our knowledge about membrane receptors, and to resolve atomic detail using methods applicable also to other integral membrane bound proteins and receptors.
- For the first time, IMAC has been used on a recombinant integral membrane protein, expressed bovine rhodopsin, at high purification factors (greater than or equal to 500) and high load (greater than or equal to 30 nmol/ml) for 5-10 mg per batch;
- 15N-lysine has been synthesized and used to produce approximately 25 mg of purified labelled rhodopsin.
- Squid opsin has been expressed in substantial quantities in Sf9 cells using a baculovirus vector;
- Doubly (or multiply) 13C and 2H labelled retinals have been prepared;
- The first deuterated retinal NMR spectra have been obtained, permitting the structure of the retinal in rhodopsin to be deduced even in the absence of the protein scaffold;
- For the first time, the dipole-dipole splitting of the signals from two 13C atoms has been directly observed allowing accurate (about +/-0.05Å) measurement of short distances to be made;
- MAS NMR of doubly labelled (11,20-13C2)-11-cis-retinal in rhodopsin has been accomplished;
- A truly European effort accomplished the study of the same retinal samples in several NMR spectrometers, at a variety to sites, to observe the effects of different field strengths on R2 spectra;
- A graphical simulation package for analysing R2 magnetisation exchange curves has been developed.
MAJOR SCIENTIFIC BREAKTHROUGHS
There were three major aspects to this project: sample production for NMR study, production of labelled retinals and amino acids, and development of the NMR methodology. This involved significant interaction between the groups, continuing beyond the contract duration, which would not have been achieved to the same extent without EC support
Protein and sample preparative methods
Exisiting expertise at Leeds, Nijmegen and Oxford was applied to produce rhodopsin for NMR studies. Bovine rhodopsin with 13C-labelled chromophore. Native bovine opsin was isolated in photoreceptor membranes in batches of 20-25 mg protein (> 90% purity on a protein basis). Batches of 50-60 mg were regenerated with 13C-labelled retinals (Nijmegen & Leiden), with a good yield (greater than or equal to 90%) at a low excess of retinal, and were analyzed by solid-state NMR (Leiden & Stockholm). To improve regeneration of native squid opsin, various conditions were tested (Nijmegen & Oxford) and an improvement was obtained from less than or equal to 5% to 25 +/- 5%, sufficient for solid-state NMR studies. Large-scale protein purification His-tagging was used in combination with immobilized metal affinity chromatography (IMAC) - an almost unique approach using an eukaryotic membrane protein. Extensive optimization trials resulted in an excellent purification factor (400-600) with good recoveries (80%) and a high column capacity (1.3 mg/ml resin). This procedure can be scaled up to 5-10 mg batches. Scale-up of recombinant mammalian opsin production. A scale-up of the regular S 400 ml suspension culture to 12 1itre, without a considerable sacrifice in production level and with a simpler infection procedure, was achieved (Nijmegen). Functional expression was also tested for squid opsin (Nijmegen & Leeds). Large-scale production of labelled rhodopsin. A protein-free synthetic media and 15N-lysine was used to produce purified labelled rhodopsin (with 50% label-incorporation). Solid-state 15N-NMR analysis will be performed shortly (Leiden) and will be the first such analysis of a labelled eukaryotic membrane protein. Other rhodopsins: Leeds has been mainly concerned with investigating the expression of cephalopod rhodopsin in eukaryotic cell systems but, after many attempts, regeneration was not successful (Leeds, Oxford & Nijmegen). Sample preparation for NMR:. Preparation of oriented membrane films using isopotential centrifugation (from Nijmegen) was used for the NMR studies (Oxford).
Retinal and lysine chemistry
The syntheses of isotopically (13C and 2H) labelled retinals have been optimized during this project. Preparation of 10-20 of 13C2retinal and 11-20 13C2 retinal in the all-E and 11-cis forms have been successful. These materials have been used for static and MAS solid state NMR measurements (Stockholm & Oxford). A large scale preparation of approximately 40g 15N2 L-lysine has been developed and used to generate expressed rhodopsin containing this amino acid. This is the first time that large scale membrane protein expression coupled with specific isotopic labelling has been possible.
Solid state NMR
The use of solid state NMR to study large integral membrane proteins within the membrane is still very much in its infancy and we have been involved in developing the methodology with some very important advances being made. The types of NMR measurements performed have been: rotational resonance (R2) measurements to estimate distances between labelled 13C sites; double-quantum-filtering experiments to isolate signals from introduced double 13C labels; and, newly developed methods to measure molecular torsional angles directly in the solid state. Prior to R2 measurements on proteins, crystalline 13C 2-labelled retinals were used to evaluated the method. An extensive study of R2 retinal line shapes was performed (Stockholm & Leiden) on four NMR instruments (4.7 T in Stockholm, 7.1 T in Bayreuth, 9.4 T in Leiden, and 14.1 T in Karlsruhe). This showed that the relatively low magnetic field in Stockholm produced R2 line shapes which behave according to the standard theoretical predictions, and so accurate internuclear distance measurements are possible from this machine. R2 measurements have now been carried out on all trans retinal crystals containing two 13C2 isotopes at 10-20 and 11-20 positions leading to the splitting of the signals caused by the dipole-dipole coupling. Also, the 1-D 13C rotational measurements have been performed on 10-20 13C2 rhodopsin, showing the dipole-dipole splitting of the signals, this will lead to accurate distance measurement between atoms in the chromophore. Experimental and theoretical studies on 1-D 13C rotational resonance NMR investigations on 13C2 labelled all-E retinal and rhodopsin have been carried out (Leiden & Stockholm).
Funding SchemeCSC - Cost-sharing contracts
6500 HD Nijmegen
2300 RA Leiden
106 91 Stockholm
LS2 9JT Leeds - West Yorkshire