Probing the Conformation and Orientation of Adsorbed Enzymes Using Side-Chain Modification

Abstract

The bioactivity of enzymes that are adsorbed on surfaces can be substantially influenced by the orientation of the enzyme on the surface and adsorption-induced changes in the enzyme’s structure. Circular dichroism (CD) is a powerful method for observing the secondary structure of proteins; however, it provides little information regarding the tertiary structure of a protein or its adsorbed orientation. In this study, we developed methods using side-chain specific chemical modification of solvent-exposed tryptophan residues to complement CD spectroscopy and bioactivity assays to provide greater detail regarding whether changes in enzyme bioactivity following adsorption are due to adsorbed orientation and/or adsorption-induced changes in the overall structure. These methods were then applied to investigate how adsorption influences the bioactivity of hen egg white lysozyme (HEWL) and glucose oxidase (GOx) on alkanethiol self-assembled monolayers (SAMs) over a range of surface chemistries. The results from these studies indicate that surface chemistry significantly influences the bioactive state of each of these enzymes, but in distinctly different ways. Changes in the bioactive state of HEWL are largely governed by its adsorbed orientation, while the bioactive state of adsorbed GOx is influenced by a combination of both adsorbed orientation and adsorption-induced changes in conformation.

Vibrational circular-dichroism spectroscopy of homologous cyclic peptides designed to fold into β helices of opposite chirality

“…The intensities of VCD peaks in these solution measurements are a factor 10 4 lower than the intensities of the corresponding IR peaks. While ECD on surfaces is well established, 19,20,22,[25][26][27]49 direct VCD measurements of peptides adsorbed on surfaces thus will be challenging and will require us to explore sensitivity enhancement strategies. Examples include increasing the absorption on surfaces, for instance, by using stacked or multilayer samples or adsorption on microparticles or nanoparticles, and we note that Bieri et al 50 and Yao et al 51 recently exploited the high surface area of metal nanoparticle suspensions to measure VCD spectra of adsorbed small molecules.…”

“…[9][10][11][12][13][14] For extending the conformational analysis to small peptides in biointerphases, we are particularly interested in optical spectroscopies because they can be employed either ex situ or in situ to obtain information-potentially in real time-about molecular conformation, chirality, and orientation. [15][16][17][18][19][20] Electronic CD ͑ECD͒, which uses circularly polarized light in the UV range and in the simplest cases provides information about the relative orientation of backbone amide chromophores, is emerging as a promising tool for investigating peptide and protein conformation on surfaces. [19][20][21][22][23][24][25][26][27] While ECD is a powerful technique for characterizing the overall conformation of chia͒ Author to whom correspondence should be addressed; electronic mail: thomas.clark@nrl.navy.mil ral biomolecules, ECD relies on transition moments that are delocalized over large portions of the molecule, resulting in broad spectral features, for which a detailed correlation with peptide conformation can be ambiguous.…”

“…[15][16][17][18][19][20] Electronic CD ͑ECD͒, which uses circularly polarized light in the UV range and in the simplest cases provides information about the relative orientation of backbone amide chromophores, is emerging as a promising tool for investigating peptide and protein conformation on surfaces. [19][20][21][22][23][24][25][26][27] While ECD is a powerful technique for characterizing the overall conformation of chia͒ Author to whom correspondence should be addressed; electronic mail: thomas.clark@nrl.navy.mil ral biomolecules, ECD relies on transition moments that are delocalized over large portions of the molecule, resulting in broad spectral features, for which a detailed correlation with peptide conformation can be ambiguous. A complementary technique, vibrational CD ͑VCD͒, uses circularly polarized IR light and provides spectra with detailed "fingerprints" analogous to standard IR spectra.…”

Experimental characterization of adsorbed protein orientation, conformation, and bioactivity

“…15,17 Some of the methods that can provide information on adsorbed protein orientation and tertiary (and quaternary) structure include fluorescence, [18][19][20][21] time-of-flight secondary-ion mass spectrometry, [22][23][24] nuclear magnetic resonance spectroscopy (NMR), 25,26 and amino acid labeling/mass spectrometry (AAL/MS). [27][28][29][30][31] Methods for the determination of secondary structure of adsorbed proteins include Fourier transform infrared spectroscopy, 32,33 surface enhanced Raman scattering, 34,35 and circular dichroism spectropolarimetry (CD). 27,[36][37][38][39] Unfortunately, as the size of the protein increases, many of the spectral signatures that are needed for tertiary structure determination using fluorescence and NMR overlap, introducing much subjectivity into the analyses, thus making it difficult to accurately interpret the configuration of the adsorbed protein.…”

“…[27][28][29][30][31] Methods for the determination of secondary structure of adsorbed proteins include Fourier transform infrared spectroscopy, 32,33 surface enhanced Raman scattering, 34,35 and circular dichroism spectropolarimetry (CD). 27,[36][37][38][39] Unfortunately, as the size of the protein increases, many of the spectral signatures that are needed for tertiary structure determination using fluorescence and NMR overlap, introducing much subjectivity into the analyses, thus making it difficult to accurately interpret the configuration of the adsorbed protein. In contrast, MS has shown great promise in characterizing the adsorbed configuration of both large and small proteins at a molecular level, especially when used with AAL.…”