New method for membrane protein engineering

April 8, 2015

March’s featured paper is "Engineering the Glutamate Transporter Homologue GltPh Using Protein Semisynthesis" published in the journal Biochemistry. The paper is published by a team from the Valiyaveetil Lab.

Neurotransmitters are chemical signals used to communicate between cells in the central nervous system. Glutamate, an amino acid, is the primary excitatory neurotransmitter in the central nervous system. After glutamate releases into the extracellular space during synaptic transmission, it must be quickly cleared from the synapse in order to reset the system.

Glutamate transporters are present in membranes of nerve and glial cells to facilitate glutamate uptake into cells and prevent toxic accumulation of glutamate in the extracellular space. Dysfunction of glutamate transporter has been implicated in diseases such as Alzheimer’s and amyotrophic lateral sclerosis.

Valiyaveetil labSearching for a methodology

“Given the physiological importance of glutamate transporters, it is not surprising that they have been extensively investigated,” said Francis Valiyaveetil, Ph.D., assistant professor of physiology and pharmacology in the OHSU School of Medicine. “But, despite a large body of work, the fundamental mechanisms of substrate recognition, ion selectivity and the coupling of substrate and ion transport are not well understood.  A major stumbling block has been the lack of a methodology for precisely engineering the transporter for informative structure-function studies.”

“We know that normal neuronal function absolutely requires precise control of glutamate levels,” said Mary Heinricher, Ph.D., assistant dean for basic research in the OHSU School of Medicine. “I chose this month’s featured paper because the Valiyaveetil lab has developed an innovative approach to synthesizing a glutamate transporter homologue that will advance mechanism research, not only for glutamate transporters but potentially for a wide range of membrane transporters.” 

A semisynthesis approach

The Valiyaveetil lab has developed a methodology for precisely engineering GltPh, an archaeal homolog of mammalian glutamate transporters, which researchers describe in their paper “Engineering the Glutamate Transporter Homologue GltPh Using Protein Semisynthesis," in the journal Biochemistry.

"Our approach, which we call 'protein semisynthesis,' involves the assembly of the GltPh transporter from a synthetic peptide and recombinant segments,” said Dr. Valiyaveetil. “The synthetic peptide can be modified as desired by using chemical synthesis for the incorporation of an almost limitless variety of unnatural amino acids or by modifications to the protein backbone and therefore provides a very precise means for engineering GltPh.”    

Semisynthesis had previously only been applied to ion channel proteins; this study is the first application to a membrane transport protein. 

Next steps

The pioneering development of the semisynthesis has set the stage for investigations to uncover the functional mechanisms that operate in GltPh. “In particular, we are very interested in deciphering the roles of the various ion binding sites that are observed in the crystal structures of GltPh,” said Dr. Valiyaveetil. “Semisynthesis is particularly useful in investigating the ion binding sites in GltPh as they show a major involvement of the protein backbone.” 

The Valiyaveetil group is also presently extending the semisynthesis methodology to other membrane transport proteins. 

Financial support for this work was provided by the NIH, a Pew Scholar Award, the American Heart Association and by OHSU.


Engineering the Glutamate Transporter Homologue GltPh Using Protein Semisynthesis
Focke PJ, Annen AW, Valiyaveetil FI.Biochemistry. 2015 Mar 3;54(8):1694-702. doi: 10.1021/bi501477y. Epub 2015 Feb 17.


Pictured above: Dr. Valiyaveetil

About the School of Medicine Paper of the Month

The OHSU School of Medicine spotlights a recently published faculty research paper each month. The goals are to describe to the public the exceptional research happening at OHSU as well as inform our faculty of the innovative work underway across the school’s departments, institutes and disciplines. The monthly paper is selected by Assistant Dean for Basic Research Mary Heinricher, Ph.D.