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.
Introduction to Lysozyme Amyloidogenesis
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).
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.
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.
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.
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.
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.
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).
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.
RESULTS: Perturbation of
Amyloidogenic Conditions of Lysozyme
- 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.
- 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).
Results: Concomitant use of Osmolytes with Amyloidogenic conditions of
Name of Individual
Techniques used with indicated
Structure of Molecule
Single Osmolyte at Alkaline pH
- Arginine analogues
- Putrescine Analogues
- Testing of fluorescence quenching by polyamines : Because Amines are quenchers of fluorescence, quenching of Thioflavin-T fluorescence by amines
- Tri Methyl Amine Oxide (TMAO)
- Ectoine and Hydroxy-Ectoine
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].
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
- 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.
Location Of Segments
Position (s) of
No. of Resi-
Alphabetic Code of Segment