• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biophysjLink to Publisher's site
Biophys J. May 2002; 82(5): 2709–2719.
PMCID: PMC1302059

Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study.


This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.

Full Text

The Full Text of this article is available as a PDF (282K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baron MH, Revault M, Servagent-Noinville S, Abadie J, Quiquampoix H. Chymotrypsin Adsorption on Montmorillonite: Enzymatic Activity and Kinetic FTIR Structural Analysis. J Colloid Interface Sci. 1999 Jun 15;214(2):319–332. [PubMed]
  • Bentley GA, Delepierre M, Dobson CM, Wedin RE, Mason SA, Poulsen FM. Exchange of individual hydrogens for a protein in a crystal and in solution. J Mol Biol. 1983 Oct 15;170(1):243–247. [PubMed]
  • Birktoft JJ, Blow DM. Structure of crystalline -chymotrypsin. V. The atomic structure of tosyl- -chymotrypsin at 2 A resolution. J Mol Biol. 1972 Jul 21;68(2):187–240. [PubMed]
  • Brady L, Brzozowski AM, Derewenda ZS, Dodson E, Dodson G, Tolley S, Turkenburg JP, Christiansen L, Huge-Jensen B, Norskov L, et al. A serine protease triad forms the catalytic centre of a triacylglycerol lipase. Nature. 1990 Feb 22;343(6260):767–770. [PubMed]
  • Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg JP, Bjorkling F, Huge-Jensen B, Patkar SA, Thim L. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature. 1991 Jun 6;351(6326):491–494. [PubMed]
  • Byler DM, Susi H. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers. 1986 Mar;25(3):469–487. [PubMed]
  • Carter DC, Ho JX. Structure of serum albumin. Adv Protein Chem. 1994;45:153–203. [PubMed]
  • Chirgadze YN, Fedorov OV, Trushina NP. Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water. Biopolymers. 1975 Apr;14(4):679–694. [PubMed]
  • Chittur KK. FTIR/ATR for protein adsorption to biomaterial surfaces. Biomaterials. 1998 Mar;19(4-5):357–369. [PubMed]
  • de Jongh HH, Goormaghtigh E, Ruysschaert JM. The different molar absorptivities of the secondary structure types in the amide I region: an attenuated total reflection infrared study on globular proteins. Anal Biochem. 1996 Nov 1;242(1):95–103. [PubMed]
  • Derewenda U, Brzozowski AM, Lawson DM, Derewenda ZS. Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase. Biochemistry. 1992 Feb 11;31(5):1532–1541. [PubMed]
  • Derewenda U, Swenson L, Green R, Wei Y, Dodson GG, Yamaguchi S, Haas MJ, Derewenda ZS. An unusual buried polar cluster in a family of fungal lipases. Nat Struct Biol. 1994 Jan;1(1):36–47. [PubMed]
  • Derewenda U, Swenson L, Green R, Wei Y, Yamaguchi S, Joerger R, Haas MJ, Derewenda ZS. Current progress in crystallographic studies of new lipases from filamentous fungi. Protein Eng. 1994 Apr;7(4):551–557. [PubMed]
  • Derewenda U, Swenson L, Wei Y, Green R, Kobos PM, Joerger R, Haas MJ, Derewenda ZS. Conformational lability of lipases observed in the absence of an oil-water interface: crystallographic studies of enzymes from the fungi Humicola lanuginosa and Rhizopus delemar. J Lipid Res. 1994 Mar;35(3):524–534. [PubMed]
  • Derewenda ZS, Derewenda U, Dodson GG. The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 A resolution. J Mol Biol. 1992 Oct 5;227(3):818–839. [PubMed]
  • DESNUELLE P, SARDA L, AILHAUD G. [Inhibition of pancreatic lipase by diethyl-p-nitrophenyl phosphate in emulation]. Biochim Biophys Acta. 1960 Jan 29;37:570–571. [PubMed]
  • Ferrato F, Carriere F, Sarda L, Verger R. A critical reevaluation of the phenomenon of interfacial activation. Methods Enzymol. 1997;286:327–347. [PubMed]
  • Goormaghtigh E, Cabiaux V, Ruysschaert JM. Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier-transform infrared spectroscopy on hydrated films. Eur J Biochem. 1990 Oct 24;193(2):409–420. [PubMed]
  • Goormaghtigh E, Cabiaux V, Ruysschaert JM. Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds. Subcell Biochem. 1994;23:329–362. [PubMed]
  • Gregory RB, Lumry R. Hydrogen-exchange evidence for distinct structural classes in globular proteins. Biopolymers. 1985 Feb;24(2):301–326. [PubMed]
  • Grochulski P, Li Y, Schrag JD, Bouthillier F, Smith P, Harrison D, Rubin B, Cygler M. Insights into interfacial activation from an open structure of Candida rugosa lipase. J Biol Chem. 1993 Jun 15;268(17):12843–12847. [PubMed]
  • Krimm S, Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv Protein Chem. 1986;38:181–364. [PubMed]
  • Lawson DM, Brzozowski AM, Rety S, Verma C, Dodson GG. Probing the nature of substrate binding in Humicola lanuginosa lipase through X-ray crystallography and intuitive modelling. Protein Eng. 1994 Apr;7(4):543–550. [PubMed]
  • Malmsten M. Formation of Adsorbed Protein Layers. J Colloid Interface Sci. 1998 Nov 15;207(2):186–199. [PubMed]
  • Martinelle M, Holmquist M, Hult K. On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. Biochim Biophys Acta. 1995 Oct 5;1258(3):272–276. [PubMed]
  • Momsen WE, Brockman HL. Recovery of monomolecular films in studies of lipolysis. Methods Enzymol. 1997;286:292–305. [PubMed]
  • Oberg KA, Fink AL. A new attenuated total reflectance Fourier transform infrared spectroscopy method for the study of proteins in solution. Anal Biochem. 1998 Feb 1;256(1):92–106. [PubMed]
  • Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag J, et al. The alpha/beta hydrolase fold. Protein Eng. 1992 Apr;5(3):197–211. [PubMed]
  • Pantazaki A, Baron MH, Revault M, Vidal-Madjar C. Characterization of Human Serum Albumin Adsorbed on a Porous Anion-Exchange Support. J Colloid Interface Sci. 1998 Nov 15;207(2):324–331. [PubMed]
  • Patton JS, Carey MC. Watching fat digestion. Science. 1979 Apr 13;204(4389):145–148. [PubMed]
  • Peters GH, Svendsen A, Langberg H, Vind J, Patkar SA, Toxvaerd S, Kinnunen PK. Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase. Biochemistry. 1998 Sep 8;37(36):12375–12383. [PubMed]
  • Prestrelski SJ, Byler DM, Thompson MP. Infrared spectroscopic discrimination between alpha- and 3(10)-helices in globular proteins. Reexamination of Amide I infrared bands of alpha-lactalbumin and their assignment to secondary structures. Int J Pept Protein Res. 1991 Jun;37(6):508–512. [PubMed]
  • Roussel A, Canaan S, Egloff MP, Rivière M, Dupuis L, Verger R, Cambillau C. Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest. J Biol Chem. 1999 Jun 11;274(24):16995–17002. [PubMed]
  • SARDA L, DESNUELLE P. Action de la lipase pancréatique sur les esters en émulsion. Biochim Biophys Acta. 1958 Dec;30(3):513–521. [PubMed]
  • Servagent-Noinville S, Revault M, Quiquampoix H, Baron M. Conformational Changes of Bovine Serum Albumin Induced by Adsorption on Different Clay Surfaces: FTIR Analysis. J Colloid Interface Sci. 2000 Jan 15;221(2):273–283. [PubMed]
  • Surewicz WK, Mantsch HH. New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochim Biophys Acta. 1988 Jan 29;952(2):115–130. [PubMed]
  • Swedberg SA, Pesek JJ, Fink AL. Attenuated total reflectance Fourier transform infrared analysis of an acyl-enzyme intermediate of alpha-chymotrypsin. Anal Biochem. 1990 Apr;186(1):153–158. [PubMed]
  • van Tilbeurgh H, Egloff MP, Martinez C, Rugani N, Verger R, Cambillau C. Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography. Nature. 1993 Apr 29;362(6423):814–820. [PubMed]
  • Wantyghem J, Baron MH, Picquart M, Lavialle F. Conformational changes of Robinia pseudoacacia lectin related to modifications of the environment: FTIR investigation. Biochemistry. 1990 Jul 17;29(28):6600–6609. [PubMed]
  • Wieloch T, Borgström B, Piéroni G, Pattus F, Verger R. Product activation of pancreatic lipase. Lipolytic enzymes as probes for lipid/water interfaces. J Biol Chem. 1982 Oct 10;257(19):11523–11528. [PubMed]
  • Winkler FK, D'Arcy A, Hunziker W. Structure of human pancreatic lipase. Nature. 1990 Feb 22;343(6260):771–774. [PubMed]
  • Zoungrana T, Findenegg GH, Norde W. Structure, Stability, and Activity of Adsorbed Enzymes. J Colloid Interface Sci. 1997 Jun 15;190(2):437–448. [PubMed]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...