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Study On The Destabilization Of Lysozyme And The Chaperone-Like Activity Of Alpha Crystallin From Sokoto Red Goat Eye Lens

Study On The Destabilization Of Lysozyme And The Chaperone-Like Activity Of Alpha Crystallin From Sokoto Red Goat Eye Lens

ABSTRACT

Destabilization of Lysozyme and chaperone like action of alpha crystallin isolated from goat’s eye lens was investigated at various temperature ranges in phosphate buffer (pH 7.1) solution and dithiothretol (DTT). This was monitored spectrophotometrically at 260nm. The heat and DTT-induced destabilization of lysozyme was prevented by alpha crystallin in a concentration dependent manner. Alpha crystallin like other chaperones, fulfils its chaperone like action in preventing aggregation of denatured proteins by the formation of complexes.

TABLE OF CONTENTS

TITLE PAGE--i
DEDICATION--ii
CERTIFICATION-iii
ACKNOWLEDGEMENTS-iv
TABLE OF CONTENTS--v
LIST OF TABLES---viii
LIST OF FIGURES--ix
ABSTRACT--x
CHAPTER ONE-1
INTRODUCTION AND LITERATURE REVIEW----1
1.0 INTRODUCTION--1
1.1.1 PROTEIN FOLDING---3
1.1.2 PROTEIN DENATURATION--5
1.1.3 CRYSTALLINS.-8
1.1.4 JUSTIFICATION-9
1.1.5 AIM AND OBJECTIVES-9
1.2 LITERATURE REVIEW--10
1.2.1 α-CRYSTALLINS: PROPERTIES, OCCURANCE AND FUNCTIONS-10
1.2.2 FUNCTIONS OF ALPHA CRYSTALLIN AND ITS RELATION SHSPS-14
1.2.3 GENE STRUCTURE, EXPRESSION AND REGULATION-15
1.2.4 QUATERNARY STRUCTURE MODELS OF CRYSTALLIN-18
vi
1.2.5 IN VITRO MODIFICATIONS OF ALPHA CRYSTALLIN--19
1.2.6 FUNCTION OF CRYSTALLIN-19
1.2.7 LYSOZYME-21
1.2.8 DITHIOTHRETOL-25
1.2.9 LENS-26
CHAPTER TWO-28
2.0 MATERIALS AND METHODS--28
2.1 MATERIALS--28
2.1.1 REAGENTS AND EQUIPMENT---28
2.1.2 APPARATUS USED FOR CHROMATOGRAPHY-28
2.2 METHODS-28
2.2.1 PREPARATION OF ALPHA CRYSTALLIN-28
2.2.2 DETERMINATION OF ALPHA CRYSTALLIN-28
2.2.3 ESTIMATION OF PROTEIN BY FOLIN-CIOCALTEU
[LOWRY] METHOD-29
2.2.4 PURIFICATION OF CRYSTALLIN USING GEL CHROMATOGRAPHY-30
2.2.5 TEMPERATURE DESTABILIZATION OF OXIDIZED LYSOZYME AND
CHAPERONE EFFECT OF ALPHA CRYSTALLIN--32
CHAPTER THREE-33
3.0 RESULTS, DISCUSSION AND CONCLUSION---33
3.1 RESULTS---33
3.2 DISCUSSION--42
3.3 CONCLUSION-47
vii
REFERENCES-48
APPENDICES--58

CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

Proteins are the workhorses of the living cell. Although proteins may differ in sequence, shape and function, but have in common, the same stereo configuration (i.e. they all have to fold into specific three-dimensional structures) which are mandatory for proper function (Bruce et al., 2002). Protein structures however are not rigid, but have a dynamic life style, which may involve unfolding and refolding, complex association and dissociation (Anfisen, 1972). Stress and also many physiological events require proteins to surrender their structure or to regain it at a later stage. A very large number of distinct conformations exist for the polypeptide chain of which a protein spends most of its time in the native conformation, which spans only an extremely small fraction of the entire configuration space. Thus, the amino acid sequence of proteins must satisfy two requirements: one, thermodynamics and the other kinetic. The thermodynamics requirement is that the sequence must have a unique folded conformation, which is stable under physiological conditions.

Most proteins can be denatured by heat, which has complex effect on the weak interactions in proteins (Vandenberg et al., 2000). If the existing temperature is increased slowly, a protein conformation generally remains intact until an abrupt loss of structure and function occurs over a narrow temperature range (Nelson and Cox, 2008). The spatial arrangement of atoms in a protein is called its conformation (Deechongkit et al., 2004).

The possible conformations of a protein include any structural state it can achieve without breaking covalent bonds (Nelson and Cox, 2008). A change in conformation could occur, by rotation about single bonds. The conformations existing under a given set of conditions are usually the ones that are thermodynamically the most stable that is, having the lowest Gibbs free energy (G). Proteins in any of their functional, folded conformations are called native protein (Ellis, 2006). The term stability refers to the tendency to maintain a native conformation. Native proteins are only marginally stable; the ΔG separating the folded and unfolded states in typical proteins under physiological conditions is in the range of only 20-65 kJ/mol. The chemical interactions that stabilize native conformation include disulfide (covalent) bonds and weak non-covalent interactions (Bruce et al., 2002).

For the intracellular proteins of most organisms, weak interactions are especially important in folding of polypeptide chains into their secondary and tertiary structures (Berg et al., 2002). The association of multiple polypeptides to form quaternary structure also relies on these weak interactions.

Secondary structure of proteins refers to any chosen segment of polypeptide chain and describes the local spatial arrangement of its main-chain atoms (Shakhnovich, 1997), A regular secondary structure occurs when each dihedral angle remains the same or nearly the same throughout the segment, The most prominent structures are alpha helix and beta conformations (Finkelstein et al., 1995). The overall three-dimensional arrangement of all atoms in a protein is referred to as the proteins tertiary structure (Sewasga & Sugihara (1984). Tertiary structure includes longer-range aspects of amino acid sequence. Amino acids that are far apart in the polypeptide sequence and are in different types of secondary structure may interact within the completely folded structure of a protein.

Some proteins contain two or more separate polypeptide chains, or subunits, which may be identical or different (Vandenberg, 2000). The arrangement of these protein subunits in three-dimensional complexes constitutes quaternary structure. Proteins are classified into fibrous and globular in this structure with polypeptide chains arranged in long strands or sheets and folded into a spherical or largely of a single type of secondary structure, and their tertiary structure (Johnson et al., 2005).

Protein quaternary structure results from interactions between the subunits of multisubunits (multimeric) proteins or large protein assemblies. Some multimeric proteins have a repeated unit consisting of a single subunit or a group of subunits, or promoter (Nelson and Cox, 2008).

1.1.1 PROTEIN FOLDING

All proteins begin their existence on a ribosome as a linear sequence of amino acid residues (Lee & Tsai, 2005). This polypeptide must fold during and following synthesis to take up its native conformation (Deechongkit et al., 2004). Although native protein is slightly stable, modest changes in the proteins environment can bring about structural changes that can affect their function. Protein folding is the physical process by which a polypeptide folds into its characteristics and functional three-dimensional structure from random coil (Alexander et. al., 2007). Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA to a linear chain of amino acids (Anfinsen, 1973). This nascent polypeptide lacks a developed three-dimensional structure. Amino acids interact with each other to produce a well-defined 3-dimensional structure, the folded protein, known as the native state. The resulting 3-dimensional structure is thus determined by the amino acid sequence (Berg et al., 2002).

The correct 3-dimensional structure is essential to the function of the protein although some parts may remain unfolded (Berg et al., 2002). Failure to fold into native structure produces inactive proteins. (Dennis, 2003).

The cell controls many of these protein folding processes itself, others are forced onto the cell from the environment. Essential processes are events such as de novo synthesis of proteins, protein translocation into different compartments, or control of the activity of regulatory proteins. A major force from outside that damages protein structures is heat stress due to increase in temperature. Similar problems can be caused by chemical compounds such as solvents or heavy metals. (Ellis, 2006). DNA damage may lead indirectly to protein folding problems because the mutated proteins are often less stable than the wild type. Protein folding defects have important medical implications. (Dennis et al., 2003). They are associated e. g. with amyloid diseases such as Alzheimer and prion diseases such as Creutzfeldt-Jakob-Disease, (Anfinsen, 1973). But they are also a major cause of cancer due to destabilising mutations in the tumour suppressor protein p53 (Bruce, et al., 2002). Likewise, several genetic disorders relate to protein folding defects: mutations in the CFTR protein that lead to its misfolding cause cystic fibrosis (Johnson, 2005).

Every cell in every organism owns an arsenal of molecular chaperones to control folding and unfolding of proteins, or to react on protein unfolding during stress conditions. Most molecular chaperones are members of evolutionary conserved families: Hsp100, Hsp90, Hsp70 and their DnaJ (Hsp40) co-chaperones, the chaperonins Hsp60, and the small heat shock proteins (sHsp). Nearly all organisms have at least one homologue of each of these classes. Severely destabilised proteins are usually degraded. Interesting is what happens to proteins that are damaged in a way that they can be kept in solution by molecular chaperons. Chaperones act as the cellular thermometer: the binding specificity of a chaperone provides the cellular definition of what is an unfolded protein. Studying the structural properties of chaperone substrates should give a direct insight about the structural properties of unfolded proteins inside the cell under physiological condition. (Shakhnovich et al., 1997).

1.1.2 PROTEIN DENATURATION

Denaturation is a conformational alteration of a biological function. This is the process by which the three-dimensional structure is lost resulting in loss of function. Most proteins can be denatured by heat, as a result of complex effects on the weak interactions in a protein (primarily hydrogen bonds). If the temperature is increased slowly, a protein’s conformation generally remains intact until an abrupt loss of structure occurs over a narrow temperature range. The abruptness of the change suggests that unfolding is a cooperative process; loss of structure in one part of the protein destabilizes other parts.

A fully denatured protein lacks both tertiary and secondary structure, and exists as a so called random coil. Under certain conditions some proteins can refold; however many cases denaturation is irreversible. Cells sometimes protect their proteins against the denaturing influence of heat with enzymes known as chaperones (Bryngelson et al.,
1995).

In quaternary structure denaturation, protein sub-units are dissociated and or the spatial arrangement of protein sub-units is disrupted. In secondary structure denaturation, proteins lose all regular repeating patterns such as alpha helices and beta pleated sheets, and adopt a random coil configuration. The amino acids sequence of primary structure held together by covalent peptide bonds is not disrupted by denaturation (Dennis, 2003).

Most proteins lose their biological function when denatured. For example, enzymes lose their activity because the substrates can no longer bind to active site and because amino acids residues involved in stabilising substrate transition states are no longer positioned to be able to do so (Dennis, 2003).

Proteins can also be denatured by extreme pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea and guanidine hydrochloride, or by detergents. Under certain conditions some proteins can refold, however, in many cases, denaturation is irreversible (Shortle, 1996). Globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity if returned to conditions in which the native conformation is stable. This process is called renaturation. (Dennis, 2003).

Thermally-induced transitions such as denaturation are detected by excess heat absorbed during the transition from the native to denatured state. Thus, the relative amounts of native and denatured protein can be determined by the magnitude of heat absorption, as demonstrated by Senisterra et al., (1997). Senisterra et al., (1997) found that the
minimum temperature for thermal induction of Hsp synthesis was quantitatively the same as the minimum temperature for denaturation of thermo labile, cellular proteins.

Essentially, who found that the minimum temperature that induced Hsp 70 transcription, approximately 40°C, represented the onset temperature (T1) or the temperature of the first endothermic transition where a specific set of thermo labile proteins unfolded due to low conformational stability, these thermo labile cellular proteins maintained conformational stability as long as the temperature was approximately 37°C (Lepock et al., 1997). Thermal stresses, i.e., rising the temperature to 40°C, caused these proteins to denature. The relationship between conformational stability of a protein and the temperature of its environment are integrally related. At physiological temperatures, a protein in its native folded state is only slightly more stable than in an unfolded conformation (Creighton, 1993). As temperature is increased, random thermal fluctuations away from the most compact conformation (Buttler and Falke, 1996) increase the probability of transition to an unfolded state or states. The conclusions of Lepock et al. (1997) was that in whole cells thermal stress caused proteins of low conformational stability to unfold, thus inducing the heat shock response (Lepock et al., 1997 and Senisterra et al., 1997).

Folding of proteins require molecular chaperones, protein at interacts with partially, folded or improperly folded polypeptides, facilitating correct folding pathways providing micro-environments in which folding can occur. The process of folding, particularly for large proteins, appears to occur via a limited number of pathways that involve distinct intermediate states [e.g. molten globule state] (Ptitsyn, 1991). These compact, partially folded intermediates transiently display hydrophobic regions on their surfaces that can interact to form insoluble aggregates (Ellis, 2006). Conditions favouring the accumulation of proteins exposing such hydrophobic residues will result in increased protein aggregation and consequently precipitation (Martin and Hartl, 1996). The unfolding path way is reversible i.e. it is possible for protein in an unfolded state to refold into the native state, provided the protein does not enter the off-folding pathway towards aggregation and precipitation (Linder et al., 1997). Molecular chaperones are family of proteins that assist in formation of the correct [native] protein structure by preventing improper or incorrect reactions that would result in protein misfolding and aggregation (Ehrnsperger et al., 1997), without becoming a part of the native structure, chaperones ensure high fidelity in protein folding and assembly in the cell, proteins from many unrelated families have recently been shown to have chaperone function (Ellis, 2006).

Chaperones are classified into two; the alpha A-crystallin and alpha B-crystallin. The alpha-crystallin account for approximately one third of the total soluble protein in the lens contributing to its refractive power (Horwitz, 1992). Alpha B-crystallin also found outside the lens having a characteristic tissue distribution. It has been shown in humans that naturally occurring point mutation in the alpha-crystallin result in deficit in chaperone-like function and can cause cataract as well as desmin related myopathy (Horwitz, 1992).


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