Abstract
Membrane transporters are a vital class of proteins for which there is little available structural and thermodynamic information. The Major Facilitator Superfamily (MFS) is a large group of transport proteins responsible for transporting a wide range of substrates in eukaryotes and prokaryotes. We have used far-UV circular dichroism (CD) to assess whether transporters from this superfamily have the same chemical and thermal stability. We have compared the stability of five different MFS transporters; PepTSo from Shewanella oneidensis and LacY, GalP, GlpT and XylE from Escherichia coli, as well as a known stable mutant of LacY, LacY-C154G. CD stability measurements revealed that these transporters fall into two broad categories. The ‘urea-sensitive’ category includes LacY-WT, GalP and GlpT, which each lose around a third of their secondary structure in 8 M urea and two-thirds in the harsher denaturant guanidine hydrochloride (GuHCl). The ‘urea-resistant’ category includes LacY-C154G, XylE and PepTSo. These resistant transporters lose very little secondary structure in 8 M urea, and LacY-C154G and PepTSo resist denaturation by GuHCl up to a concentration of 4 M. The stabilities of LacY, GlpT, XylE and PepTSo correlated with their crystal structure conformations, implying that a similar conformation is adopted in vitro. The ‘urea-sensitive’ transporters LacY and GlpT were crystallised inward-open states, while XylE and PepTSo were crystallised in occluded states. This study highlights the importance of studying a wide range of similar proteins, as a similar secondary structure and overall function does not necessarily confer the same stability in vitro.
Highlights
The Major Facilitator Superfamily is a large group of secondary transporters found in all organisms, responsible for transporting a wide range of substrates (Yan 2015)
To ascertain whether other Major Facilitator Superfamily (MFS) transporters exhibit the same behaviour, the transporters GlpT, PepTSo, LacY-C154G and XylE were each unfolded in urea, and measured by circular dichroism (CD) to assess the loss of secondary structure (Figs. 2, 3)
The six MFS transporters studied here fall into two broad categories, the ‘urea-sensitive’ including LacY-WT, GalP and GlpT, and ‘urea-resistant’, including XylE, LacYC154G and PepTSo
Summary
The Major Facilitator Superfamily is a large group of secondary transporters found in all organisms, responsible for transporting a wide range of substrates (Yan 2015). By using CD to compare the stability of different transporters of the MFS, we can directly compare the secondary structure changes induced upon denaturation without the difficulties in interpretation which can arise from other techniques such as fluorescence. Despite the similar structure of the MFS transporters studied, there are significant differences in stability to chemical denaturant between the different transporters, indicating that behaviour in vitro cannot be generalised across a whole family of proteins. This highlights the need to study as many membrane proteins as possible, in order to find the factors that govern stability in vitro and facilitate the study of transporters with a role in health and disease. LacY measurements with 10 mM lactose was measured at a protein concentration of 0.3 mg ml−1 and a cell pathlength of 0.2 mm
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