Structural Biochemistry

 

Previous Research Results:

We are interested on the molecular mechanisms of transport and regulation in ion transport membrane proteins. Our studies are focused on a couple of potassium channels, both from eukaryotic and prokaryotic organisms, one ion symporter and an ion antiporter.  In these studies we combine X-ray crystallography, for three-dimensional structure determination, with different biochemical and biophysical techniques.

The MlotiK1 cyclic nucleotide regulated potassium channel is one of the proteins which we have been studying. This channel is activated upon binding of cAMP just like the CNG and HCN channels in humans. In the past few years, together with the lab of Lise Thomas in the US, we have characterized the functional, structural and biochemical properties of this potassium channel. In particular, we have determined the structure of the full-length channel as well as several different structures of its regulatory domain. We now understand fairly well the molecular changes that occur at the level of the regulatory domain when it binds or unbinds cAMP. Currently, we are developing a molecular model for the molecular changes which occur at the level of the channel during gating. For this purpose, and together with Daniel Müller in Switzerland, we have recently characterized the conformational transition of the channel using Atomic Force Microscopy.

 

IMAGE: Side-view of the cyclic nucleotide regulated potassium channel.

 

Future Research:

At present one of our aims is the expansion of the set of technical tools used in our studies. We have started using fluorescence spectroscopy to characterize the mechanism of the membrane proteins. In particular, we have been developing ion transport assays using fluorescence probes that are sensitive to K+ concentration. We are now hoping to start using FRET techniques in the cell to characterize large multiprotein complexes that include the channels we have been studying.

 

Selected references:

Clayton G, Silverman W, Heginbotham L, Morais Cabral JH (2004) “Structural basis of ligand activation in a cyclic nucleotide regulated potas­sium channel. ” Cell 119:615-27.

Albright RA, Vazquez Ibar JL, Kim CU,Gruner SM, Morais-Cabral JH (2006) “The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring.” Cell 126: 1147-59

Albright RA. Joh K, Morais-Cabral JH (2007) “Probing the structure of the dimeric KtrB membrane protein.” Journal of Biological Chemistry 282: 35046-55

Clayton GM, Altieri S, Heginbotham L, Unger VM, Morais-Cabral JH (2008) “Structure of the transmembrane regions of a bacterial cyclic nucleotide-regulated channel.” PNAS U S A. 105:1511-5.

Clayton GM, Aller SG, Wang J, Unger V, Morais-Cabral JH. “Combining electron crystallography and X-ray crystallography to study the MlotiK1 cyclic nucleotide-regulated potassium channel” J. Structural Biol. (2009), 167: 220-6.

Ricardo Simão Vieira-Pires and João Henrique Morais-Cabral “310 helices in channels and other membrane proteins” J. Gen. Physiol. (2010), 136:585-592

 

 

 

Group Leader
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jcabral@ibmc.up.pt
People
Researchers

Fernandes, Andreia
afernand@ibmc.up.pt

Harley, Carol
harleyca@ibmc.up.pt

Martinez, Omar Santin
omar.santin@ibmc.up.pt

PosDocs

Jorge, João
joao.jorge@i3s.up.pt

Rocha, Rita
rita.rocha@ibmc.up.pt

Phd Students

Barros Reis Cereija, Tatiana
tatiana.cereija@ibmc.up.pt

Duarte, Celso
celso.duarte@ibmc.up.pt

Collaborators

Hryshkina, Anastasiya
hryshkina@ibmc.up.pt


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