植物非專一性脂質運輸蛋白第二型為一可溶於水溶液的蛋白質,且已被證實其在活體外(in vitro)具有運輸脂質於膜間的功能。此蛋白質具有八個高度保留性的半胱胺酸所形成的四對雙硫鍵以穩定此蛋白質結構。在植物非專一性脂質運輸蛋白的三度空間立體結構中，其內部具有一斥水性空腔，為用來與受質結合的重要功能部位，其可與脂肪酸，磷脂質，及固醇類等斥水性或斥水親水兩性分子結合，並協助它們於膜間運送。雖然植物非專一性脂質運輸蛋白質第二型的結構早已被發表，但關於其受質結合與脂質運送功能的詳細特性與機制都尚未清楚。在這篇研究中，我們率先使用點突變的技術製作一系列點突變的非專一性脂質運輸蛋白質，以用來闡明不同的斥水性胺基酸對於蛋白質結構、穩定性、受質結合能力與脂質傳輸能力的重要程度。我們的結果證明三個點突變脂質輸送蛋白質L8A、F36A，和V49A幾乎失去了原本完整的三級結構，並且因而失去了其受質結合能力與脂質輸送能力。除此之外，芳香族胺基酸的側鏈對於斥水性結合裝載作用(hydrophobic packing)十分地重要。我們將蛋白質序列位置45與48上的兩個酪胺酸分別進行點突變成丙胺酸以來驗證此兩個芳香族胺基酸對於此蛋白質的影響為何。與原本的蛋白質做比較，結果發現此兩個點突變蛋白質在結構上並沒有顯著改變。然而，其受質結合能力與脂質運輸能力卻都明顯地下降。這顯示此兩個酪胺酸對於其蛋白質的功能扮演很重要的角色。另外要特別指出的是，點突變F39A蛋白質不只增加蛋白質螺旋程度，還顯著地增加其磷脂質結合能力與脂質運輸能力，雖然其麥角固醇(ergosterol)結合能力下降。單一點突變將蛋白質序列上的苯丙胺酸39突變成丙胺酸後，改變了蛋白質的原本結構，且影響了其受質結合與運輸的機制。 這一系列的點突變蛋白研究對於”非專一性脂質輸送蛋白質第二型的結構與功能關係”提供了非常顯著且重要的資訊。

Plant nonspecific lipid transfer protein 2 (nsLTP2) is a soluble protein which is characterized by its ability to transfer phospholipids between membranes in vitro. Eight highly conserved cysteine residues can form four disulfide bonds to stabilize the folded conformation of nsLTP2. The three dimensional structure of nsLTP2 also reveals an inner hydrophobic cavity which may serves as a potential binding site for the hydrophobic or amphiphilic ligands such as fatty acids, phospholipids, and sterols. Although the structures of nsLTP2 have been reported before, the detailed properties/mechanisms of the ligand binding and lipid transfer activity are still unclear. In this study, a series of nsLTP2 mutants were made by site-directed mutagenesis to elucidate the importance of various hydrophobic residues on protein structure, stability, ligand binding and lipid transfer activity. Our results demonstrate that three individual mutations (L8A, F36A, and V49A) almost destroy the native tertiary structure of nsLTP2 and therefore lose its ability in ligand binding and lipid transfer. Besides, the aromatic side chains are usually critical in the hydrophobic packing. Two mutations in the tyrosine residues, Y45A and Y48A, were generated to exam their roles in nsLTP2. There is no obvious structural change in these two singly mutant proteins comparing with wild type protein. However, the ligand binding and lipid transfer activity are apparently decreased. This is suggested that these two residues play a significant role in biological activity. And especially point out that the F39A mutant not only increases the helical content, but also significantly enhances the phospholipid binding and lipid transfer activity even though the ergosterol binding ability was decreased. A change of Phe39 to Ala alters the conformation of nsLTP2, which may affect the binding and transfer mechanism. These mutants have provided significant information about the structure-to-function relationships of nsLTP2.

1. Rueckert, D.G. and Schmidt, K. (1990) Lipid transfer proteins. Chem. Phys. Lipids 56: 1-20.
2. Kader, J. C. (1996) Lipid-transfer proteins in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 627-54.
3. Han, G. W., Lee, J. Y., Song, H. K., Chang, C., Min, K., Moon,J., Shin, D. H., Kopka, M. L., Sawaya, M. R., Yuan, H. S., Kim,T. D., Choe, J., Lim, D., Moon, H. J., and Suh, S. W. (2001) Structural basis of non-specific lipid binding in maize lipid-transfer protein complexes revealed by high-resolution X-ray crystallography, J. Mol. Biol. 308, 263-78.
4. Lerche, M. H., Kragelund, B. B., Bech, L. M., and Poulsen, F.M. (1997) Barley lipid-transfer protein complexed with palmitoylCoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands, Structure 5,291-306.
5. Sodano, P., Caille, A., Sy, D., de Person, G., Marion, D., and Ptak, M. (1997) 1H NMR and fluorescence studies of the complexation of DMPG by wheat non-specific lipid transfer protein. Global fold of the complex. FEBS Lett. 416: 130-4.
6. Jegou, S., Douliez, J. P., Molle, D., Boivin, P., and Marion, D. (2000) Purification and structural characterization of LTP1 polypeptides from beer, J. Agric. Food Chem. 48, 5023-9.
7. Douliez, J. P., Pato, C., Rabesona, H., Molle, D., and Marion, D. (2001) Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2, Eur. J. Biochem. 268, 1400-3.
8. Samuel, D., Liu, Y. J., Cheng, C. S., and Lyu, P. C. (2002) Solution structure of plant nonspecific lipid transfer protein-2 from rice(Oryza sativa), J. Biol. Chem. 277, 35267-73.
9. Pons, J. L., de Lamotte, F., Gautier, M. F., and Delsuc, M. A. (2003) Refined solution structure of a liganded type 2 wheat nonspecific lipid transfer protein, J. Biol. Chem. 278, 14249-56.
10. Douliez, J. P., Michon, T., K., and Marion, D. (2000) Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels, J. Cereal Sci. 32, 1-20.
11. Chao-Sheng Cheng, Dharmaraj Samuel, Yaw-Jen Liu, Je-Chyi Shyu, Szu-Ming Lai, Ku-Feng Lin, and Ping-Chiang Lyu. (2004) Binding mechanism of nonspecific lipid transfer proteins and their role in plant defense, Biochemistry. 43, 13628-13636
12. Lindorff-Larsen, K., and Winther, J. R. (2001) Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases, FEBS Lett. 488, 145-8.
13. Liu, Y. J., Samuel, D., Lin, C. H., and Lyu, P. C. (2002) Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa), Biochem. Biophys. Res. Commun. 294, 535-40.
14. Yu YG, Chung CH, Fowler A, Suh SW. Amino acid sequence of a probable amylase/protease inhibitor from rice seeds. Arch Biochem Biophys 1988;265:466–75.
15. Zhou HX, Lyu P, Wemmer DE, Kallenbach NR. Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: evidence for an N terminal “capping box”. Proteins 1994;18:1–7.
16. Greene, R. F., Jr., and Pace, C. N. (1974) Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, alphachymotrypsin, and beta-lactoglobulin, J. Biol. Chem. 249, 5388-93.
17. Santoro, M. M., and Bolen, D. W. (1988) Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha- chymotrypsin using different denaturants, Biochemistry 27, 8063-8.
18. Goddard, T. D., and Kneller, D. G. (1999) SPARKY 3, University of California, San Francisco.
19. Mikes, V., Milat, M. L., Ponchet, M., Panabieres, F., Ricci, P., and Blein, J. P. (1998) Elicitins, proteinaceous elicitors of plant defense, are a new class of sterol carrier proteins, Biochem. Biophys. Res. Commun. 245, 133-9.
20. Douliez JP, Michon T, Marion D. (2000) Steady-state tyrosine fluorescence to study the lipid binding properties of a wheat non-specific lipid transfer protein (nsLTP1). Biochim Biophys Acta 1467, 65–72.
21. Guerbette F, Grosbois M, Jolliot-Croquin A, Kader JC, Zachowski A. (1999) Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants. Biochemistry 38, 14131–7.
22. Van Paridon, P. A., Gadella, T. W., Jr., Somerharju, P. J., and Wirtz, K. W.(1988) Biochemistry 27, 6208–6214
23. Cheng, C. S., Chen, M.N., Liu, Y.J., Huang, L.Y., Lin, K. F.,and Lyu, P.C.(2004) Evaluation of plant non-specific lipid-transfer proteins for potential application in drug delivery. Enzyme and Microbial Technology 35,532–539.
24. Kamal, J. K., and Behere, D. V. (2002) Thermal and conformational stability of seed coat soybean peroxidase, Biochemistry 41, 9034-42.
25. Greene, R. F., Jr., and Pace, C. N. (1974) Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, alphachymotrypsin, and beta-lactoglobulin, J. Biol. Chem. 249, 5388-93.
26. Santoro, M. M., and Bolen, D. W. (1988) Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha- chymotrypsin using different denaturants, Biochemistry 27, 8063-8.
27. Wallace, A.C., Laskowski, R.A., and Thornton, J.M. (1995). LIGPLOT: A program to generate schematic diagrams of protein–ligand interactions. Protein. Eng. 8, 127–134.
28. Pato C, Le Borgne M, Le Baut G, Le Pape P, Marion D, Douliez JP. (2001) Potential application of plant lipid transfer proteins for drug delivery. Biochem Pharmacol. 62, 555–60.
29. Avery L. McIntosh, Adalberto M. Gallegos, Barbara P. Atshaves, Stephen M. Storey, Deepak Kannoju, and Friedhelm Schroeder. (2003) Fluorescence and Multiphoton Imaging Resolve Unique Structural Forms of Sterol in Membranes of Living Cells. J. Biol. Chem., Vol. 278, 8, 6384-6403.
30. Lyu PC, Liff MI, Marky LA, Kallenbach NR. (1990) Side chain contributions to the stability of alpha-helical structure in peptides. Science., 250(4981):669-73.