A Thesis Submitted towards Partial Fulfilment of the Requirements for the Degree of

I hereby declare that the matter embodied in this thesis, entitled: “Disruption of amyloidogenic disorders of Hen Lysozyme, inhibition thereof by naturally occurring osmolytes and numerical code of amino acids” is the result of research carried out by me in the Department of Biosciences & Bio-engineering, Indian Institute of Technology Guwahati, India under the supervision of Prof Rajaram Swaminathan. Nividh Chandra for the award of the degree of Doctor of Philosophy is an authentic account of the results obtained from the research work carried out under my supervision in the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, India.

Introduction to Lysozyme Amyloidogenesis

Introduction to Interaction of Osmolytes with Proteins

Experimental Methodology used in this Thesis

Results: Perturbations of Amyloidogenic Conditions of Lysozyme

Numeric code of Amino Acids and its Applications

Discussion & Future Directions

Four Important Abbreviations

These Abbreviations Are Important because they have been used Extensively in this Thesis (especially to document the

Other Abbreviations Used in This Thesis

25.rhGCSF Recombinant human granulocyt colonstimulerende factor 26.rhIL-1RA Recombinant human interleukin-1 receptor antagonist 27.rhIFN-y Recombinant human interferongamma.

Abstract of Thesis

In Chapter 6 of the thesis, a new numerical code of amino acids is proposed for visual use. This numerical code has been applied to the transmembrane tendency of amino acids in the protein Rhodopsin.

Another advantage of the numerical code is that it helps with easy memorization and recall of the primary and secondary structure of a small protein.

Chapter-1

Introduction to Lysozyme Amyloidogenesis

PROTEIN AGGREGATION

Intrinsic factors include different structural levels of the protein: primary, secondary, tertiary and quaternary. Changes in protein structure during protein unfolding or refolding under stress conditions lead to conformational changes that can cause aggregation.

Mechanisms of protein aggregation: For any given protein, there can be multiple aggregation pathways, and each pathway can involve multiple intermediate steps

In a broad sense, protein aggregation refers to the process by which protein molecules tend to adopt non-native conformations in adverse environmental conditions and polymerize to form stable complexes of various sizes, called protein aggregates. The rate and extent of aggregation is influenced by numerous factors that are categorized as extrinsic and intrinsic factors.

1.111 Aggregation through unfolding protein intermediate

1.112 Aggregation through self-association or chemical linkages (Physical aggregation)

Cross-linking of protein chains can also lead to protein aggregation involving intermolecular disulfide bond formation/exchange, formaldehyde-mediated cross-linking, di-tyrosine formation, oxidation and Maillard-type reactions. Disulfide bond formation or exchange between surface-located cysteines is the most common chemical bond leading to protein aggregation.

1.113 Chemical degradation-induced protein aggregation

PROTEIN AGGREGATION: Mostly unwanted
Diseases associated with protein aggregation – Aggregation of some physiologically important proteins such as βamyloid- peptides, insulin, lysozyme and many
LYSOZYME AGGREGATION and AMYLOIDOGENESIS 1 Systemic amyloidosis in human lysozyme
Human and Hen Lysozyme structure similarity
Hen lysozyme aggregation previous literature

Spino-cerebral ataxia: SCA is also caused by polyglutamine expansion in the ataxin-3 protein, resulting in protein aggregation. However, protein aggregation is not limited to neuropathies but also causes some systemic disorders (Soto et al, 2001).

Table 1.1: Examples of different proteins and their aggregation mechanisms 4

Chapter-2

Introduction to Interaction of Osmolytes with Proteins

Physiological occurrence and Biological roles of osmolytes

Many different osmolytes can occur even in the same organism or cell, and can also replace each other in times of shortage. They also accumulate the organic osmolytes available in the environment, namely trehalose, proline, ectoine, glycine betaine and carnitine, of which betaine and proline are best accumulated by intake.

Table 2.3: Physiological occurrence of osmolytes in different organisms (Burg and Ferraris, 2008)

HOW OSMOLYTES INFLUENCE PROTEIN AGGREGATION?

Different osmolytes have different impact on the protein aggregation -
ARGININE: Arginine is one of the most extensively studied osmolyte against protein aggregation. It is a positively charged amino acid with a complex
TREHALOSE: Trehalose is a non-reducing disaccharide of glucose which is essential in many organisms like yeasts, plants and insects for its abilities to

LEA PROTEINS and TREHALOSE 23 : Late embryogenesis abundant (LEA) protein initially identified in plants are now documented in animals also. These are extremely

TMAO: TMAO is a widely present in marine animals as an osmotic pressure regulator in cell cytoplasm and is known to counteract the effect of urea in ratio
GLYCINE BETAINE: GB is the stress counteracting solute which acts as osmoprotectant in both prokaryotic and eukaryotic cells. It is a quaternary
PROLINE: Proline is an osmoprotectant which inhibits aggregation of proteins in vivo as well as in vitro. The mechanism of the stabilizing nature of proline
GLYCEROL: Glycerol is one of the most widely used polyols against protein aggregation in formulation of biopharmaceutics and in food industry. It affects
SORBITOL: Sorbitol follows same mechanism of preferential hydration as other polyols like glycerol but it is a better stabilizer than glycerol 41 . The
TAURINE: Taurine is a free amino acid abundantly found in mammalian cells

Direct interaction of the osmolyte with the amino acid side chains on the protein surface, destabilizing the protein. GB slows down the aggregation of tetra-cys CRABP protein, by destabilizing the intermediate monomeric forms of the protein 22.

Table 2.4: Different Osmolytes and their Effect on Proteins

CONCLUSION

FUTURE PERSPECTIVE and CHALLENGES

However, this aspect still remains to be worked on, because oral administration of sugars results in their degradation into their respective monomers and absorption in the small intestine. Based on experimental studies, various mechanisms of suppression or enhancement of aggregation caused by osmolytes have been proposed, but the exact molecular basis of the phenomenon is still lacking. The lack of understanding is due to the disparity in the functional behavior of osmolytes with different proteins.

Mechanism for benzyl alcohol-induced aggregation of recombinant human interleukin-1 receptor antagonist in aqueous solution. Effects of pH, temperature, and sucrose on benzyl alcohol-induced aggregation of recombinant human granulocyte colony-stimulating factor. Mechanism for preventing protein aggregation: a case study of α-globulin aggregation in glycerol.

Thesis Objectives

This has been shown that at acidic pH/elevated temperature, lysozyme begins to break up into pieces 1. This was done at a single time point, but it can be expected that multiple time points would give more overlapping pieces of increasingly smaller size. When the idea first started, we found that it is not only useful for remembering the primary sequence, but also has applications in other domains of the 'Amino Acid Scale'.

Studying them all with many more techniques and with more protein and at more time points would yield so much data that would be difficult to assimilate when presented with pictures. It became clear at the beginning of my experimental work with Lysozyme that I would work exclusively on this protein during the entire tenure of my PhD. As a curiosity, it occurred to me that if I'm going to spend so many years on a single protein that isn't very long, why not memorize its primary sequence and secondary structure.

Chapter-3

Materials used

All osmolytes (at least 98% pure), HEWL (95% pure, the rest are buffer salts), human lysozyme, thioflavin-T, and Micrococcus lysodeikticus [as a powder] were purchased from Sigma-Aldrich. Sodium dihydrogen phosphate dihydrate, potassium chloride, sodium bicarbonate, sodium hydroxide and DMF were purchased from Merck Limited, Mumbai and were of GR grade.

Buffers

HEWL stocks

Preparation of Osmolyte Stocks and Final Reaction Mixtures for Alkaline and Acidic Conditions

Samples for Ethanol condition

It is worth noting that visual acuity for adults is ∼30 µm] and remains suspended in Brownian motion, thus appearing homogeneous and monophasic. Although far from the ideal solution, it is nevertheless a solution, and therefore extremely well-developed principles of solution-state physical chemistry can be applied [and many techniques that work on the solution state can be used]. Once this is done and results in amyloidogenesis at higher temperatures, we can use the same and it is likely that many osmolytes will dissolve at such high temperatures.

Guandinium Hydrochloride Samples

Heating and Cooling of Samples: All 25 o C (alkaline pH and ethanol) samples were kept in an incubator with both heating and cooling mechanism activated

General Methodology used in this thesis with Rationale

Mechanism of Lysozyme Action
Enzyme Activity as a measure of extent of Folded Lysozyme
Observations and Concerns We Have
Measurement of Residual Enzymatic Activity

Figure-3.2 Part of the polysaccharide component of bacterial cell walls showing the alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues. First, a nearby aspartate residue (Asp 52), which is in the negatively charged carboxylate form, interacts with the positive charge of the oxonium ion. Lytic activity of hen egg white lysozyme as a function of solution ionic strength at pH 6.2, 8.0 and 9.2.

Thioflavin-T Fluorescence for measuring extent of Amyloid

Even though the recommended pH of the Thioflavin-T test was 8.5, when we added a sample with pH 12.2, the final pH became 10.9, which is well above the recommended value and still showed fluorescence enhancement at 482 nm. Nevertheless, we believe that this approach can be a satisfying exercise and provide valuable input into the mechanism of Thioflavin-T. The emission intensity at 482 nm [which was the maxima of this graph] was recorded [after subtracting the buffer-only blank].

Theory of Steady State Anisotropy (ss-Anisotropy) Experiments

Protein Labelling with Dansyl dye

IHV/IHH gives G factor (which is the ratio of the sensitivities of the detection system to vertically and horizontally polarized light) which is calculated during the course of measurements. Here η is viscosity, ̅ is specific volume of the protein (volume per gram of protein) which is typically 0.73 ml/g and h is hydration which is typically 0.23 g H2O per gram of protein. Although Dansyl Chloride is believed to react with Lysine side chains (there are six Lysine residues in HEWL).

Figure 3.12: Mechanism of Dansyl Chloride Reaction with Proteins

The abbreviations used in next chapters to denote results from various techniques is as under

Insights into the pathway of hen egg white lysozyme amyloid fibril formation obtained from a small-angle X-ray and neutron scattering study. Interaction of thioflavin T with synthetic Alzheimer's beta-amyloid peptides: detection of amyloid aggregation in solution. Slow aggregation of lysozyme at alkaline pH monitored in real time using fluorescence anisotropy of a covalently labeled dansyl probe.

Chapter-4

RESULTS: Perturbation of

Amyloidogenic Conditions of Lysozyme

Alkaline pH

HEWL and Human Lysozyme behave similarly
Perturbation of pH
Effect of pH on HEWL REA
Effect of pH on r ss of dansyl-labelled HEWL
Effect of pH on Thioflavin T fluorescence (measured after 10 days)
Effect of sample temperature on HEWL REA
Effect of sample temperature on r ss of dansyl-labelled HEWL
Effect of sample temperature on Thioflavin-T fluorescence
Effect of HEWL concentration on REA
Effect of seeding
A. Effect of seeding observed using r ss of dansyl-labelled HEWL
B. Effect of seeding on Thioflavin T fluorescence
Dimerization of HEWL at different pH observed using r ss

Figure-4.4: REA (slope of first 30 s) of HEWL 120 µM incubated in pH 12.2 at different temperatures at different time intervals. Figure-4.5: Steady state anisotropy of Dansyl conjugated HEWL (118 + 2) incubated at pH 12.2 at different temperatures for different time intervals. Figure-4.8: Steady state anisotropy of dansyl-conjugated HEWL incubated at pH 12.2 in 25oC for various times as indicated.

Figure 4.4 shows the effect of sample incubation temperature on the activity of HEWL exposed to pH 12.2

Effect of temperature jump on Thioflavin T fluorescence
Lateral addition of dansyl-labelled HEWL

How this experiment was carried out?
Conclusions from this experiment

Ethanol Condition

REA of 120 µM HEWL incubated at indicated times and indicated concentrations of ETHANOL in water (v/v)
Steady State Anisotropy of dansyl-labelled HEWL in presence of Ethanol
Light scattering from HEWL samples in presence of Ethanol
Thioflavin-T of HEWL samples incubated at Ethanol condition

Acidic pH
Guanidinium Condition

Figure-4.13 shows the effect of different concentrations of ethanol on the enzymatic activity of HEWL (120 µM). Figure-4.13: REA of 120 µM HEWL incubated at indicated concentration of ethanol (v/v) in water for indicated number of days. Figure-4.16: 120 µM HEWL incubated at indicated concentrations of ethanol in water (V/V) for 1-month sample (top) and 1-year sample (bottom).

Figure 4.18: REA of 600 µM HEWL incubated at pH 2.0 at 57 o C

Chapter-5

Results: Concomitant use of Osmolytes with Amyloidogenic conditions of

Lysozyme

Osmolyte

Generic Name

Name of Individual

Techniques used with indicated

Molecule

Structure of Molecule

Single Osmolyte at Alkaline pH

L-Arginine
Arginine analogues
Polyamines
Putrescine Analogues
Testing of fluorescence quenching by polyamines : Because Amines are quenchers of fluorescence, quenching of Thioflavin-T fluorescence by amines
Trehalose
Tri Methyl Amine Oxide (TMAO)
Ectoine and Hydroxy-Ectoine
Taurine
Betaine

Figure: 5.2: REA HEWL 120 µM incubated at pH 12.2 at 25 C (for all samples) with different concentrations of ARGININE shown. Figure-5.7: ThT HEWL (120 µM) at pH 12.2 and 25oC incubation for indicated days and indicated for arginine analogues [concentration of arginine and its analogues is 500mM]. Figure-5.8: Th-T HEWL (120 µM) at pH 12.2 and incubated at 25 oC for the indicated concentrations with arginine and argininamide [recorded after 5 days].

Figure 5.2 shows the influence of increasing arginine concentration on the residual enzymatic activity (REA) of HEWL measured at different times since incubation in alkaline pH (12.2)

Mixture of Osmolytes (under Alkaline Condition)

Recently an article was published describing the synergistic effect of two osmolytes (Proline & Sorbitol) as an inhibitor of the aggregation of Lysozyme & Insulin 10. In the future we want to study combinations of more than four osmolytes, but if we combine more osmolytes use, so many combinations may arise that they will be difficult to study in a single experiment, especially time-consuming experiments such as residual enzymatic activity. One experiment is presented using cytoplasm (especially from extremophiles) instead of a mixture of osmolytes.

Osmolytes at acidic pH condition

However, they used only two osmolytes and only one amyloidogenic state of lysozyme (ie, the guandinium hydrochloride state). However, as the following table shows, the reaction mixtures did not behave as well as under alkaline conditions. In the alkaline condition for all osmolytes [even at very high concentrations] the fluid was homogeneous, free of any particles and free flowing [not viscous] and so from the beginning forever.

Osmolyte Physical appearance of the sample reaction mixture

Control Trehalose- 500mM

Conclusions

Facile memorization of primary and secondary structure of proteins
B,C etc. in the following refers to Species as given below
Sus scrofa (wild boar, Eurasian wild pig)
Rattus norvegicus (brown rat, common rat, street rat, sewer rat) C) Mus musculus (house mouse, laboratory mouse)
Pan troglodytes (common chimpanzee) E) Homo sapiens (Human)
Danio rerio (Zebrafish) G) Gallus gallus (Chicken)
Visual Bioinformatics Analysis

It is expected that the entire primary and secondary structure (especially for shorter peptide) can be memorized quickly and with excellent recall after long periods of time. Nevertheless, we are on the threshold of happiness that we have 20-AAs, so the remaining 10 can be considered underline from 0-9. Strings can be memorized in short sequences which is often the case with spools and beta sheets.