Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein Journal Article
| Authors: | Kenoth, R.; Zou, X.; Simanshu, D. K.; Pike, H. M.; Malinina, L.; Patel, D. J.; Brown, R. E.; Kamlekar, R. K. |
| Article Title: | Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein |
| Abstract: | HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2W208F, HET-C2W208A and HET-C2F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λmax by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λmax blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λmax blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λmax blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar. © 2018 Elsevier B.V. |
| Keywords: | fluorescence; membrane binding; glycosphingolipid transfer; gltp-fold; gltp; het-c2; trp point mutation |
| Journal Title: | Biochimica et Biophysica Acta (BBA) - Biomembranes |
| Volume: | 1860 |
| Issue: | 5 |
| ISSN: | 0005-2736 |
| Publisher: | Elsevier B.V. |
| Date Published: | 2018-05-01 |
| Start Page: | 1069 |
| End Page: | 1076 |
| Language: | English |
| DOI: | 10.1016/j.bbamem.2018.01.001 |
| PROVIDER: | scopus |
| PUBMED: | 29305831 |
| PMCID: | PMC5963984 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 March 2018 -- Source: Scopus |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work