Biophysical analysis of glycated proteins

Abstract

Abstract for thesis part 1 – Protein Glycation Analysis

Glycation is a non-enzymatic post-translational modification that occurs between free amino acid groups of proteins or peptides and carbohydrates. This process affects the structure, function and stability of proteins and is accelerated under conditions like hyperglycaemia. The resulting advanced glycation endproducts (AGEs) have implications in many age-related chronic diseases. Numerous analytical methods are currently employed for studying these protein modifications. In this part of the thesis, I will be reporting the developments of a novel gel electrophoresis based affinity method for the detection and analysis of protein glycation. A variety of proteins, including the most abundant circulating protein, human serum albumin (HSA), are examined along with more complex samples such as human serum and material from an Alzheimer’s Disease model.

Our laboratory has previously shown that incorporation of a specialised carbohydrate affinity ligand, methacrylamido phenylboronic acid (MPBA), in polyacrylamide gels enabled an improved separation of carbohydrates. In this thesis, the development of a boronate assisted SDS-PAGE electrophoresis method (mP-AGE) will be described that facilitates the separation of glycated proteins. This technique not only separates glycated from unmodified proteins, it also differentiates between different glycation states, and distinguishes glycation products resulting from reactions with different sugars, such as glucose (glucation) and fructose (fructation). In addition, I describe further improvements of the mP-AGE technique in order to enhance the separation between glycated and unmodified proteins by utilising boronic acid gradients, carbohydrate imprinting, and novel 2D gel techniques. Finally, I present a new method to visualise glycated proteins in complex samples prior to protein staining by using fluorescent boronic acid based carbohydrate ligands. The results presented in this thesis show that mP-AGE is a cheap yet versatile methodology for the analysis of glycated proteins. This technique may aid the development of new proteomics tools for the detection and separation of glycated proteins.

Abstract for thesis part 2 – Crystallographic analysis of proteins

Staphylococcus aureus is a gram positive bacterium that causes a range of illnesses, ranging from minor skin infections to life-threatening diseases. It secretes a number of soluble proteins that aid the evasion of the complement pathway, which is the first line of defence against microbial infections. One of these proteins, Sbi (Staphylococcus aureus binder of IgG), interacts with components of both innate and adaptive immune systems of the host. The proposed extracellular region of Sbi consists of four domains, two IgG binding domains (Sbi-I and II) and two additional domains (Sbi-III and IV). Sbi-IV interacts with complement C3 and thus inhibits the alternative pathway. However, in the presence of domain III (Sbi-III-IV), it induces futile consumption of complement component C3. Resolving the structure of complement inhibitory molecules facilitates the development of therapeutics for auto-immune diseases. Previous work in our laboratory showed two binding modes in the crystal structure between Sbi-IV and C3d. One interaction site is located on the concave face of C3d, a binding site that is shared by other Staphylococcal immune proteins, and another on the convex side of the molecule. In this section of the thesis I will be describing the expression, purification and crystallisation of Sbi III-IV in complex with C3d. Sbi-III-IV was co-crystallised with C3d to gain understanding of the role of Sbi domain III. However, the protein was cleaved during crystallisation and co-crystals of Sbi-IV and C3d were obtained in a crystal form that was markedly different from the previously published structure. In this part of my thesis, the interactions between Sbi-IV and C3d in the two crystal forms will be examined.

Date of Award	1 Sept 2011
Original language	English
Awarding Institution	University of Bath
Supervisor	Jean Van Den Elsen (Supervisor)