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Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein
| Content Provider | Scilit |
|---|---|
| Author | Kenoth, Roopa Zou, Xianqiong Simanshu, Dhirendra K. Pike, Helen M. Malinina, Lucy Patel, Dinshaw J. Brown, Rhoderick E. Kamlekar, Ravi Kanth |
| Copyright Year | 2018 |
| Description | Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes 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 $HET-C2^{W208F}$, $HET-C2^{W208A}$ and $HET-C2^{F149Y}$ all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas $HET-C2^{F149A}$ 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 $HET-C2^{W109Y/F149Y}$ 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 $λ_{max}$ by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and $λ_{max}$ 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 $λ_{max}$ blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp $λ_{max}$ 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. |
| Related Links | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963984/pdf |
| Ending Page | 1076 |
| Page Count | 8 |
| Starting Page | 1069 |
| ISSN | 00052736 |
| DOI | 10.1016/j.bbamem.2018.01.001 |
| Journal | Biochimica et Biophysica Acta (BBA) - Biomembranes |
| Issue Number | 5 |
| Volume Number | 1860 |
| Language | English |
| Publisher | Elsevier BV |
| Publisher Date | 2018-01-03 |
| Access Restriction | Open |
| Subject Keyword | Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes Biochemistry and Molecular Biology Trp Point Mutation Glycosphingolipid Transfer |
| Content Type | Text |
| Resource Type | Article |
| Subject | Cell Biology Biochemistry Biophysics |
