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Hydrogen–Deuterium Exchange in Bovine Serum Albumin Protein Monitored by Fourier Transform Infrared Spectroscopy, Part I: Structural Studies

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Abstract:

The structure of the amide I band of bovine serum albumin (BSA) was determined using an H/D exchange experiment. The difference between the dry and hydrated exchange spectrum revealed the fine structure of the amide I band. The band at 1717 ± 2 cm−1 is due to the vibration of the COOH moieties from the protein side chains. Band components at 1682 ± 2 cm−1, 1655 ± 2 cm−1, and 1637 ± 2 cm−1 are assigned to the vibrations of the backbone C=O. These three bands belong to vibrations of three different populations of amide groups differing in the number of established H-bonds. The connectivity between the frequencies of various amide vibrations was determined by two-dimensional generalized correlation spectroscopy and spectral decomposition. About 7% of the whole exchangeable hydrogen atom population (NH, NH2, and OH groups from backbone and side chains) remains unexchanged, and these hydrogen atoms belong mainly to the NH groups, which are H-bonded to specific C=O groups. Moreover, this study concerns the approximately 10% of hydrogen atoms belonging to a particular HN···O=C population with a characteristic amide A frequency at 3290 cm−1 and an amide I band at 1655 ± 2 cm−1, usually attributed to the α-helical structure that remains unexchanged. At higher temperature the exchange is more efficient. Upon heating, a further 4% of these NH groups are deuterated. The comparison of the exchange spectrum at higher temperature with the structural changes of the protein at the same temperature implies that the change in overall dynamics of the protein improve the level of exchange.

Keywords: BOVINE SERUM ALBUMIN; FOURIER TRANSFORM INFRARED SPECTROSCOPY; FT-IR SPECTROSCOPY; HYDROGEN-DEUTERIUM EXCHANGE; SECONDARY STRUCTURE; SPECTRAL DECOMPOSITION; TWO-DIMENSIONAL GENERALIZED CORRELATION SPECTROSCOPY

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370205774783115

Affiliations: 1: National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia 2: Département de Recherche Fondamentale sur la Matiére Condensée, SI3M/PCM, CEA Grenoble, 17 rue des Martyrs, F 38054 Grenoble Cedex 9, France

Publication date: November 1, 2005

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