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D. Chakraborty, L. Gwyn, S. Kim, K. Rodgers, M. I. Naash; The Conformational Properties of the Second Intracellular D2 Loop of the Tetraspanin Protein RDS. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5434.
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Vertebrate photoreceptors contain a unique tetraspanin protein known as retinal degeneration slow (RDS). Mutations in the Rds gene have been identified in a variety of human retinal degenerative diseases and more than 70% of these mutations are located in the second intradiscal (D2) loop, highlighting the importance of this region. Here we examined the secondary structure of the D2 loop of RDS.
A PCR product of RDS D2 loop (Phe120 - Asn256) was subcloned into the multiple cloning sites of pMal-C2 vector to generate a MBP (Maltose Binding Protein) fusion protein. MBP-D2 was over-expressed in E. coli BL21 (DE3) and was purified using an amylose affinity column and resolved on SDS-PAGE to check the purity. Circular dichroism (CD) spectroscopy was performed on purified MBP-D2 using a JASCO J715 spectropolarimeter with a PTC-348WI peltier temperature controller (Jasco, Corp.; Tokyo, Japan).
The conformational properties of RDS D2 loop were determined using the CDpro software package. MBP-D2 and MBP each gave CD spectra typical of proteins containing primarily -helix. Contrary to the CD spectra of the two proteins, the differential CD spectrum, which should represent the spectrum for the D2 part was a mixture of β-sheet and random coil. Next, we will study the structure of the D2 loop in the absence of MBP. We are also in the process of introducing several diseases-causing mutations in the D2 loop to evaluate their effect on the structural conformation of the domain.
These results demonstrate that a purified soluble form of D2 loop of RDS takes the form of a mixture of random coil and β-sheet conformation. Evaluation of the structural properties of disease-causing RDS mutations (e.g R172W, C214S, N244H/K) that are located within the D2 domain will provide valuable insight into the mechanisms underlying Rds-associated retinal degenerations.
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