NMR Analysis of Synthetic Human Serum Albumin α-Helix 28 Identifies Structural Distortion upon Amadori Modification

Abstract

The non-enzymatic reaction between reducing sugars and long-lived proteins in vivo results in the formation of glycation and advanced glycation end products, which alter the properties of proteins including charge, helicity, and their tendency to aggregate. Such protein modifications are linked with various pathologies associated with the general aging process such as Alzheimer disease and the long-term complications of diabetes. Although it has been suggested that glycation and advanced glycation end products altered protein structure and helicity, little structural data and information currently exist on whether or not glycation does indeed influence or change local protein secondary structure. We have addressed this problem using a model helical peptide system containing a di-lysine motif derived from human serum albumin. We have shown that, in the presence of 50 mM glucose and at 37°C, one of the lysine residues in the di-lysine motif within this peptide is preferentially glycated. Using NMR analysis, we have confirmed that the synthetic peptide constituting this helix does indeed form a ␣-helix in solution in the presence of 30% trifluoroethanol. Glycation of the model peptide resulted in the distortion of the ␣-helix, forcing the region of the helix around the site of glycation to adopt a 3 10 helical structure. This is the first reported evidence that glycation can influence or change local protein secondary structure. The implications and biological significance of such structural changes on protein function are discussed.

Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices

“…Further, K 97 is part of a di-lysine motif and it is generally accepted that these are more susceptible to glycation due to local acid-base catalysis making one residue more nucelophilic. 36 The fact that K 97 was least reactive in the freeze-dried samples investigated here suggests that the local structural environment, accessibility, and charge of the lysine residues is different to that found in solution and crystal forms. Further studies to elucidate such differences between the solution and glassy state are therefore required and we note that previous studies in solution using peptide models have shown that the local environment does influence the rate of glycation and local secondary structure elements on glycation.…”

“…Also, the linking of formulation to activity is not common. 5,11,36 There was an obvious change in lytic activity only in those lysozyme samples formulated in pH 6.2 buffer and heated for 24 h at 90 C (Table 2). However, here we have shown that while changes in activity are not always measurable (Table 2), there may be chemical modification to the protein (Table 3) occurring that could potentially lead to changes in the antigenicity of the protein.…”

Structural Mechanism of Ring-opening Reaction of Glucose by Human Serum Albumin

“…1a) is an often used model protein to study the pyranose ring-opening reaction of glucose and protein glycation by many techniques, notably mass spectrometry, to obtain information about glycation site (16,18,29,30). To understand the mechanism and the structural determinant for glycation, we determined the crystal structure of rHSA in complex with glucose.…”

Role of advanced glycation end products in cardiovascular disease

“…The influence of such modifications on protein structure has been far less investigated. Recently, using nuclear magnetic resonance (NMR), Howard et al [28] have showed that glycation of a model helical peptide from human serum albumin (HSA) by glucose caused distortion of the helical structure of the protein at the point of glycation. Another protein structural study for the changes induced by non-enzymatic glycation in the secondary and tertiary protein structure of HSA showed that the in general protein structure was unaltered.…”