Conformational Changes and Orientation of Humicola lanuginosa Lipase on a Solid Hydrophobic Surface: An in Situ Interface Fourier Transform Infrared-Attenuated Total Reflection Study

Abstract

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.

The differential influence of colocalized and segregated dual protein signals on neurite outgrowth on surfaces

“…More intriguing was the finding that that these patterned laminin layers contained a more than 9-fold greater density of AL-4 recognizable epitopes than layers produced from laminin coated channels. We propose that the observed differences between μFN patterned laminin layers can be explained by differential adsorption rates [26][27][28][29], in which laminin interactions with microchannel adsorbed aggrecan molecules resulted in slower adsorption rates to glass and more favorable laminin packing in the adsorbate layer. Laminin epitope presentation was also affected by μCP, which resulted in the disruption of nearly all native AL-4 targeted epitopes.…”

“…Laminin epitope presentation was also affected by μCP, which resulted in the disruption of nearly all native AL-4 targeted epitopes. The laminin denaturation observed in stamped layers was most likely caused by strong non-specific adsorption to the hydrophobic PDMS stamps [28] and by the physical nature of the stamping process.…”

Relating material surface heterogeneity to protein adsorption: the effect of annealing of micro-contact-printed OTS patterns

“…It has been shown by others that albumins denature to a greater extent on more hydrophobic surfaces [6,42] and that initial binding rate increases with the ability of HSA to denature upon adsorption [43]. We postulate that strong hydrophobic interactions between a more denselypacked OTS region cause HSA molecules to denature upon adsorption in such a way that multiple surface binding sites are occupied.…”

Frozen Microemulsions for MAPLE Immobilization of Lipase

“…Infrared spectroscopy is a well-established technique for the conformational analysis of proteins [21] and has been used to study the secondary structure of proteins both in water solution [22,23] and in the solid state [24,25]. FTIR spectra of the three MAPLE-deposited samples, together with those of free-CRL are reported in figures 1 (a) (1300-1800 cm -1 range) and 1 (b) (2600-4000 cm -1 range), since the absorption of the various secondary structural elements of the protein backbone can be studied in these spectral regions.…”