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Contortions of membrane protein captured in new study

Researchers in the Biochemistry and Physics departments have applied an old technique to a new problem to reveal detailed information about how a protein's structure changes as it carries out its function.

The study on a bacterial ion channel, which was carried out in a fraction of the time it would take to probe the structure using conventional approaches, shows that the technique could be widely applicable to many other membrane proteins.

Reprinted from Structure, Cell Press. Copyright © 2010, with permission from Elsevier

Reprinted from Structure, Cell Press. Copyright © 2010, with permission from Elsevier

Dr Catherine Vénien-Bryan in the Department of Biochemistry and Dr Stephen Tucker in the Department of Physics collaborated with researchers in the States to gain access to the specialised equipment necessary for the technique. Together, the group has published its findings in Structure1.

The protein which the group studied, KirBac3.1, is a bacterial potassium channel which regulates the selective flow of potassium ions across the cell membrane. These channels are found in all organisms, from bacteria to humans, and are important for maintaining the cell's correct ionic balance.

Potassium channels behave as gated pores. When triggered by a variety of stimuli, the channel's shape changes, and the part which acts as a gate opens. This allows potassium ions, but no other ions, to pass through the channel, or 'permeation pathway', and into the cell's interior. The change of shape of the protein is therefore crucial for its function.

'The big question with these potassium channels is what their open and closed conformations are and how they move between them,' says Dr Tucker. ' For that you need structures. There are lots of potassium channel structures available, but there are very few that are in both states.'

"Our study shows just how powerful this relatively simple technique is - it can give an awful lot of information"

The most widely used approach to studying protein structure is 3D X-ray crystallography. After many years of effort, researchers have very recently had some success with this method. But a problem that hinders this approach is the difficulty of getting truly open or closed states of the protein.

Some years ago, Dr Vénien-Bryan embarked on using a different approach to understand the gating process. She showed that she could use cryo-electron microscopy to obtain 2D snapshots of KirBac3.1 in the open and closed states. Her work revealed that the protein underwent a large conformational change when shifting between the two states.

But producing 3D models of the channel from these structures is a time-consuming task. So Dr Vénien-Bryan was interested to discover that there was a different, potentially promising approach called radiolytic footprinting available at the National Synchrotron light source at Brookhaven, New York.

'The opportunity to do this footprinting work at Brookhaven was a chance to identify the amino acids residues that move during the gating process,' says Dr Vénien-Bryan, enabling the group to get the heart of the structural changes taking place.

In radiolytic footprinting, the protein is pulsed with X-rays which interact with the surrounding water. This produces chemical compounds that react with those parts of the protein exposed on the surface. The protein is then chopped up with an enzyme and run through a machine called a mass spectrometer which analyses the fragments, detecting which parts of the protein have been modified.

'The idea is that if the channel is closed, some residues on the protein will not be accessible, but when it opens, these residues suddenly become much more accessible,' explains Dr Tucker. 'So they will be modified much more in the open state than in the closed state. And it's not just residues that line the permeation pathway that are modified, but also crevices that open up.'

The researchers used conditions in which the protein was trapped in one or other of the two conformations and passed these samples on to their collaborators at the synchrotron facility at Brookhaven for analysis.

Mapping the modified residues. The colour codes indicate the changes in rates of modification on transition from the closed to the open state

Mapping the modified residues. The colour codes indicate the changes in rates of modification on transition from the closed to the open state

By being able to compare the open and closed state directly, the researchers have come up with an impressively detailed map of precisely which amino acids of KirBac3.1 move during gating. They identified a number of regions of the protein which move - some very significantly, others less so - including an area near the opening of the channel which controls the size of ions allowed through.

Since the channels are similar across a wide range of organisms, any studies on bacterial channels are thought to be relevant to mammalian ones. It is therefore likely that the detailed model proposed by the group will help explain how mammalian channels work.

The work also highlights the importance of this technical approach. 'Our study shows just how powerful this relatively simple technique is - it can give an awful lot of information,' comments Dr Tucker. 'It could be applied to other membrane proteins which undergo conformational change and which are very difficult to study by traditional X-ray crystallography.'

Researchers may also be able to adapt the technique so that they can probe the structural changes in ion channels in real time with millisecond resolution and potentially see the intermediate steps in the gating process.

  1. Gupta, S., Bavro, V.N., D'Mello, R., Tucker, S.T., Vénien-Bryan, C. and Chance, M.R. Conformational changes during gating of a potassium channel revealed by structural mass spectrometry. Structure 2010 Jul 14;18(7): 839-46

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