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Academic year: 2021

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In questa tesi viene presentato lo studio di due proteine amiloidi, l’insulina e l’α-sinucleina , condotto attraverso l’utilizzo della spettroscopia infrarossa a trasformata di Fourier in alta pressione. Con il nome di fibrille amiloidi ci si riferisce ad aggregati proteici altamente ordinati che si vengono a de- positare in diversi organi o tessuti durante lo sviluppo di molte importanti patologie, le amiloidosi. Tra queste, vista la crescente diffusione nelle popo- lazioni occidentali, vale la pena citare il morbo di Parkinson, e il morbo di Alzheimer. Le conoscenze relative alla termodinamica degli aggregati amiloi- di e alle cinetiche di aggregazione sono ancora limitate. Ci´o che maggiormente complica la ricerca di strategie atte ad inibire la reazione `e la sostanziale irre- versibilit´a del processo di aggregazione. Le fibrille amiloidi dimostrano infatti una sorprendente stabilit´a termodinamica. L’introduzione di tecniche di alta pressione nello studio dei sistemi biologici ha negli ultimi anni evidenziato le potenzialit´a e l’utilit´a della biofisica delle alte pressioni in particolare nello studio degli stati conformazionali delle proteine . Attraverso una variazione di pressione, si possono infatti gestire le distanze intra- e inter-molecolari in modo controllato. Dal presente studio emerge che l’applicazione dell’al- ta pressione `e in grado di indurre la rottura degli aggregati amiloidi con un’efficienza che dipende dallo stato di maturazione della fibrilla (nel caso dell’insulina) e dalla specifica mutazione genetica (nel caso della sinucleina).

E’ stato inoltre individuata una sequenzialit´a nel processo di dissociazione in- dotto, che sembra riflettere la pre-esistente gerarchia di strutture della catena peptidica. Attraverso l’applicazione dell’alta pressione `e stato dunque pos- sibile stabilizzare stati intermedi di fibrillazione. Quest’ultimo rappresenta il punto di forza della biofisica in alta pressione, che permette di popolare e quindi caratterizzare stati energeticamente instabili a pressione ambiente, fornendo dunque nuove linee di ricerca da seguire per la comprensione delle amiloidosi.



In this thesis is presented the investigation of the infrared properties at high pressures of two amyloidogenic proteins: insulin and α-synuclein. Amyloid fibrils are highly ordered protein aggregates occurring during the develop- ment of many serious diseases, like Alzheimer’s and Parkinson’s diseases.

Despite a major effort both from the biological and biophysical communi- ties, very little is known about the thermodynamics of the aggregated pro- tein state and the kinetic mechanisms of its formation. Fibrillation is an irreversible process and a key challenge is the development of therapeutical strategies able to inhibit or reverse the formation of amyloids. Hydrostatic pressure has proven to be a powerful tool for the study of biological systems.

Contrary to temperature - whose effects show both on volume and thermal energy simultaneously - pressure leads to a controlled change of inter- and intramolecular distances, and enables to determine the stability of a protein structure. From the present study, it comes out that high pressure is able to induce the disaggregation of fibrils with an efficacy which depends on the maturation stage (insulin) and on specific point mutations (synuclein). Our results show how high pressure disaggregation occurs following a sequential mechanism that reflects the protein’s structure hierarchy. Pressure allows to address intermediate states, which are probably occurring along the aggre- gation reaction pathway, thus providing a new clue to the understanding of amyloidosis.



The theme of aggregation processes in protein misfolding represents one of the most suggestive and challenging topics in the current biophysical re- search. Since a large and still growing number of human diseases has been linked to the formation of proteinaceus deposits, the study of misfolding is nowadays of crucial interest in biological sciences. Abnormal accumulation in organs of highly ordered aggregates, the so called amyloids fibrils, have been found in Parkinson’s and Alzheimer’s affected patients and so far, more than 20 other pathologies have been found to share the formation of amyloids dur- ing the development of the disease. Aggregation represents a serious problem also in industrial production of protein preparations for pharmaceutical pur- poses. The biological activity of a protein in an aggregate is in fact usually greatly reduced. More importantly, non-native protein aggregates can cause adverse reaction in patients, ranging from immune response to anaphylactic shock and even death. A significative example is represented by insulin ag- gregation, that is a serious problem for diabetic insulin dependent patients since it can lead to an injection form of amyloidosis. Thus, a major goal of formulation engineering is to design a preparation in which aggregates is kept at extremely low level. Achieving this goal in an aqueous formulations is a daunting challenge, which is further exacerbated by the current lacunae in the understanding of the protein aggregate state. Even though many studies addressed the investigation of the reaction that leads to the formation of amyloids, relatively little is known about the triggering processes and about the structure of the species participating to the reaction. To this purpose, the



research on amyloids has recently focused on the investigation of oligomers of misfolded proteins, thought to be on pathway kinetic intermediates of reaction. A large number of experimental evidences of on-pathway interme- diates of aggregation of several amyloid proteins have been reported in the last years. Whether the toxic specie is the amyloid aggregated form or the oligomeric intermediate is an open and widely debated question, central in the current research scenario. The idea of the existence of toxic intermediates suggests a protective role of amyloid deposits. The research of intermediates has been complicated by their thermodynamically and kinetically unstable nature, that did not allow an accurate characterization of their structure.

Conversely, amyloid fibrils show a surprising thermodynamic stability, mak- ing fibrillogenesis a highly irreversible reaction. Moreover, relatively little is known about amyloids structure since the difficulty in obtaining well diffract- ing crystals has hindered their X-ray diffraction characterization.

Several mechanisms are involved in protein aggregation, from protein confor- mational changes, to nucleation and growth of different kind of aggregates.

The above mechanisms involve multiple interactions arising from both direct and solvent-induced forces which are equally effective at inter and intra- molecular level. In this thesis, we will present an infrared study of high pressure dissociation of amyloid fibrils. The coupling of conventional FT- IR and high pressure techniques has proven to be a perfect combination for the study of proteins aggregation and misfolding, allowing to explore pro- teins conformational states not accessible at ambient pressure. Contrary to temperature - whose effects show both on volume and thermal energy simul- taneously - pressure leads to a controlled change of inter- and intramolecular distances, and enables to determine the stability of a protein conformation through a selective tuning of weak interactions. According to Le Chatelier’s principle, a pressure increase will favour all that processes that lead to a volume reduction, thus proteins monomerization and aggregates dissociation are expected to be favoured at high pressure. The dissociation of amyloids at high pressure enables to probe the role of electrostatic and hydrophobic


interactions as driving forces of aggregation and moreover, since intermediate states are stabilized at high pressure high pressure represents a unique tool for their characterization.

The 1st chapter of this thesis is divided into two parts. In the first part, the problem of proteins aggregation in the context of amyloidosis will be pre- sented together with the most supported models proposed in the last years for amyloid structure. The second part of the 1st chapter will be dedicated to high pressure bioscience. The attention will be focused on the application of high pressure techniques to proteins conformational tuning. A great care will be dedicated to the description of the potentiality of high pressure in the dissociation of aggregated structures.

In Chapter 2 is described high pressure Fourier transform infrared spec- troscopy (HP-FTIR) technique and the diamond anvil cell will be presented.

The experimental HP-FTIR set-up and SISSI infrared beamline of Sincrotrone Trieste will be also described. A section will be dedicated to the protocols used for sample preparation, high pressure cell loading and FT-IR measure- ments.

In Chapter 3 we will discuss the results obtained on bovine pancreatic in- sulin under pressure. We will present the high pressure behaviour of the physiological assemblies of the protein (hexameric, dimeric and monomeric) discussing the protection role played by physiological assembly against de- naturation. We will discuss the relation between high pressure stability and intermolecular packing, evaluated from the high pressure behaviour of heat induced BPI fibrils at three distinct maturation stages. The high pressure stabilization of an intermediate protofibrillar state is also discussed. More- over the high pressure crystallization of misfolded insulin is also presented.

In Chapter 4 we will present the high pressure dissociation of α-synuclein amyloids. Three fibrillar species have been studied, wild type, A30P and A53T mutants. It will be discussed the connection between the sequence dependent amyloidogenicity of the protein and the high pressure behaviour of the amyloids it builds up. High pressure dissociation will be compared to


pH dissociation, showing that the two methods are not equivalent.

Finally, in Chapter 5 some conclusion about the experimental evidences pre- sented and discussed in Chapter 3 and 4 will be drawn.

In this thesis two Appendices are also present. In Appendix A, the concept of protein structure in its hierarchic organization will be described. In Ap- pendix B, will be presented the detail of 2D-Correlation analysis applied to FT-IR spectroscopy.



Introduction iii

1 High pressure on amyloid proteins 1

1.1 Proteins misfolding . . . 1

1.1.1 The configurational landscape of proteins . . . 1

1.1.2 The misfolded state: From native to amyloids . . . 5

1.2 High pressure bioscience . . . 16

1.2.1 High Pressure Principles . . . 17

1.2.2 High Pressure stability of Proteins: elastic and plastic effects . . . 18

2 Experimental 27 2.1 Infrared Spectroscopy . . . 27

2.1.1 FT-IR principles . . . 28

2.1.2 The Absorbance . . . 31

2.1.3 FTIR on proteins . . . 32

2.1.4 High Pressure effects on proteins vibrational modes . . 38

2.2 Experimental apparatus . . . 39

2.2.1 Interferometer . . . 39

2.2.2 The infrared beamline at Sincrotrone Trieste . . . 40

2.2.3 High Pressure Diamond Anvil Cell . . . 42

2.2.4 High Pressure Infrared Setup . . . 45

2.2.5 Fluorescence system for pressure calibration . . . 46

2.3 Materials and methods . . . 48 vii


2.3.1 Diamond Anvil Cell loading . . . 48 2.3.2 Sample preparation protocols . . . 49 2.3.3 FTIR measurements . . . 50 3 Insulin as a model protein for studying fibrillogenesys 57 3.1 Physiological and pathological structural states of Insulin . . . 57 3.2 Huge resistance of insulin native assemblies to high pressure . 60 3.2.1 State of the art . . . 60 3.2.2 FTIR measurements on Hexameric Insulin in solution . 64 3.2.3 FTIR measurements on Hexameric Insulin powder . . . 66 3.2.4 FTIR measurements of insulin in acidic solution . . . . 69 3.2.5 FTIR measurements of insulin in alcoholic solution . . 72 3.2.6 Discussion . . . 75 3.3 High Pressure perturbation of the stability of insulin fibrils . . 78 3.3.1 State of the art . . . 78 3.3.2 FTIR measurements of heat induced insulin fibrils . . . 80 3.3.3 Insulin crystallization . . . 89 3.3.4 Discussion . . . 94 4 Probing the high pressure stability of misfolded α-synuclein103 4.1 The role of α-synuclein in Parkinson’s disease . . . 103 4.2 Synuclein fibrils stability: state of the art . . . 105 4.3 FTIR measurements on α-synuclein fibrils . . . 107

4.3.1 High pressure FTIR measurements on Wild Type α- synuclein . . . 108 4.3.2 High pressure measurements on A30P and A53T α-

synuclein mutant . . . 116 4.3.3 High Pressure FTIR measurements on Wild Type α-

synuclein at high pH . . . 126 4.4 Discussion . . . 131

Conclusions 139


A Proteins Structure and involved interactions 143

A.1 Primary Structure . . . 144

A.2 Secondary Structure . . . 145

A.3 Tertiary Structure . . . 148

A.3.1 Interactions stabilizing tertiary structure . . . 148

B 2D-Correlation Analysis of FTIR spectra 153 B.1 2D-Correlation Analysis principles . . . 153

B.2 Formal Description of 2D-Correlation . . . 155

B.3 The synchronous spectrum . . . 156

B.4 The asynchronous spectrum . . . 158


Chapter 1

High pressure on amyloid proteins

In this chapter we present the problem of proteins aggregation in the con- text of amyloids connected diseases. The presentation of high pressure bio- science and its application on proteins conformational studies will follow, focusing the attention in particular to high pressure stabilization of interme- diate misfolded states and amyloids disaggregation.

1.1 Proteins misfolding

1.1.1 The configurational landscape of proteins

Proteins are linear, unbranched polymers of amino acid residues that undergo, in their physiological environment (mainly composed of water) a reversible disorder ↔ order transition called protein folding (see Figure 1.1).

The natural three-dimensional arrangement, the folded native structure, is strictly connected to the function the protein accomplishes. A detailed de- scription of proteins structure can be found in Appendix A. Water is the major constituent of cells and a remarkable solvent whose chemical and phys- ical properties affect almost every aspect of life. Due to the amphipathic and charge properties of amino acids, the achievement of the native functional



Figure 1.1: Schematic representation of protein folding.

structure of a protein is reached through the best deal between H-bonds, electrostatic interactions, van der Waals forces and hydrophobic effects, able to minimize the free energy of the molecule. Many of proteins properties are thus a direct reflection of the fact that water molecules, that are the main environmental constituent, are in contact with proteins surface. Under suitable conditions, all of the information needed to realize the ordered form of most proteins is encoded in their linear sequence; no auxiliary compo- nents are necessary to guide the disordered chain to its unique, biologically relevant three-dimensional structure[1]. However, the molecular mechanism responsible for self-assembly remains an open issue, probably the most fun- damental open question in biophysics. Protein folding may sometimes fail, leading to the formation of misfolded proteins and aggregates. However, the cell has developed a protection mechanism against incorrect folding of pro- teins assigning the task to check and correct incorrect folding to particular proteins, named molecular chaperones. Unfortunately, this mechanism not always works so that proteins can undergo structural changes that destabilize the energetic balance, driving the peptidic chain towards non functional, mis- folded states. When intermolecular interactions have the best on intramolec- ular forces (that drive the protein towards its native state) aggregation may occur. The same physical forces that determine the conformations along the folding pathway, including the folded and unfolded structures, also domi-


nate the non-covalent mutual interactions between two protein molecules, proteins and other macromolecules, and proteins and solvent (a detailed de- scription of these interactions can be found in Ref. [2]). From this point of view, proteins folding and aggregation represent two sides of the same coin.

Electrostatic interactions of point charges crucially affect most long-range in- teractions of proteins with other proteins and other charged macromolecules.

The main contributors to the stabilization of globular proteins are van der Waals interactions, followed by hydrogen bond and hydrophobic interactions of non-polar residues. In order to produce a stable folded conformation, these contributions have to overcompensate the destabilizing contributions from the hydration of polar residues and the gain in conformational entropy upon unfolding. A delicate balance between stabilizing and destabilizing con- tributions causes a stability of most globular proteins in water in the range of only 10-70 kJ mol−1[3]. The ”native” conformation, resulting from folding process, depends both on the intrinsic properties of the amino-acid sequence and on multiple contributions from the environment. The native structure is a thermodynamically stable, essentially unique three-dimensional arrange- ment, characterised by the ability to fluctuate through a large number of free energy minima (conformational sub-states). The folded molecule ex- hibits a large variety of motions, like local fluctuations of single atoms or structural rearrangements of wide extent, ranging in a large variety of time scales. This flexibility is connected to disorder and molecular mobility, and make hydrated proteins classifiable among glass-forming systems, even if the substantial uniqueness of the native functional state, and the rapid folding process, distinguish proteins from truly frustrated systems. The stability and the folding pathway of a protein can be described by using a free energy landscape (see Figure 1.2). Each point on the surface in Figure 1.2 describes a specific conformation of the protein. The shape of the energy landscape is affected by the contribution of enthalpic terms due to amino acid’s interac- tions and to both enthalpic and entropic terms due to the interaction with the environment. In a simplified description, protein stability depends on


Figure 1.2: Three-dimensional representation of Protein Folding Energy Land- scape from Ref. [4]; While multiple pathways are available for the unfolded state, the native conformation is unique and corresponds to the energy minimum N, reached toward the progressive reduction of accessible conformations. It is worth to note that due to the roughness of the surface, kinetic traps are present.

free energy changes (∆G) expressed by equation 1.1:

∆G = ∆H − T ∆S (1.1)

where the enthalpic term (∆H) takes into account for the ”binding energy”

(electrostatic interactions, hydrogen bonding, van der Waals interactions), while the hydrophobic interactions are described mainly by entropy(S) driven processes. The ”folding funnel” landscape allows the protein to fold into the native state through any of a large number of pathways and intermediates.

Random fluctuations in the unfolded or partially folded states drive the re- action as different native as well as non-native contacts are sampled. In the energy landscape perspective it is necessary to phenomenologically admit a funnel-like landscape. In this context, native interactions between residues are assumed to be more stable than non-native contacts: as such contacts are formed, the number of available conformations is reduced, driving the polypeptide chain towards the deep minimum of the energy landscape. The surface of this folding funnel is unique for a specific polypeptide sequence


under a given set of conditions and it is determined by both thermodynamic and kinetic properties of the folding polypeptide chain[4][5]. The protein searches for a set of critical contacts unify the description of protein folding and aggregation. Indeed, a number of evidences state that several proteins favour intermolecular contacts with neighbouring proteins at the expenses of native contacts. In this situation, aggregated states result thermodynam- ically similar or more favourable than the native state. The driving forces toward the equilibrium state are both ”non-specific” e.g. hydrophobic and electrostatic, and specific e.g. salt-bridging between residues, and in this description, are common both to folding and aggregation[6]. The latter pro- cess can be considered as acting in competition with the normal folding pathway[7][8]. While exploring the funnel surface, local energetic minima can be encountered; they are kinetic traps slowing down the folding process, the correspondent configurations are intermediates, partially folded confor- mations, that can be intrinsically prone to aggregation[9][10]. Such states may be mandatory for the folding process, i.e. the folding protein must nec- essarily pass through these conformations in order to reach the native form, while other minima and correspondent conformations may lie away from a

”correct” pathway.

When partially folded states are significantly populated, intermediate species may interact specifically with each other to form aggregates[12][13](see Figure 1.3).

1.1.2 The misfolded state: From native to amyloids

Protein aggregation is an important phenomenon that alternatively is part of the normal functioning of nature or, central to this thesis, are linked to pathological states.

Proteins aggregation also represents a limit for protein preparation used as pharmaceutics. During the development of several important diseases proteins have been found to undergo irreversible structural changes, leading to the formation of highly ordered aggregates, the amyloid fibrils (see Figure


Figure 1.3: Free energy landscape of wild-type β-2 microglobulin, here reported as an example of multi protein energy landscape(from Ref. [11]). The free energy surface is represented as a function of two reaction coordinates, φ1 and φ2, in- dicating the non native intramolecular interactions during folding and non-native intermolecular interactions during aggregation, respectively. Local minima rep- resent the ensembles of unfolded and intermediate states. A deep minimum is assigned to the native state N, while F indicates the thermodynamically most stable structure, of the protein within an amyloid fibril.

1.4). So far more than 20 proteins have been associated to the development of a pathological state. The most diffused amyloid associated diseases are reported in Table 1.1 (the proteins studied in the present thesis are reported in bold).

Alzheimer’s disease (AD) and other neurodegenerative disorders like Parkin- son’s disease, belong to the family of such pathologies that goes under the generic name of amyloidosis due to the presence of insoluble protein aggre- gates located near the site of organs damage. These ’amyloid’ aggregates share some physico-chemical features: fibrillar morphology, secondary struc-


Figure 1.4: SEM and cryo-SEM micrographs of amyloids of:(a)Aβ1-40, related to Alzheimer’s;(b)Lisozime, related to systemic amyloidosis,(c)(all-S)SAA1-12, re- lated to secondary systemic amyloidosis;(d)(all-R) SAA1-12 from Ref. [14].

ture, insolubility in common solvents and detergents, and protease-resistance.


The polymerization of amyloidogenic proteins, yielding well-ordered fib- rils, is characterized by multiple transitional species: initial seeds, soluble small oligomers, protofibrils and insoluble polymers, amyloid fibrils. Fibril formation is a polymerization process whose kinetics can be described by a sigmoid curve (see Figure 1.5). Recently it has been suggested to occur through a complex wire of structural changes between many conformational species that can be summarized in three main steps: protein misfolding, oligomeric nucleation, and fibril elongation (see Figure 1.6). In this phe- nomenon a key role is played by hydrophobic interactions, backbone hydro- gen bonding, and stacking interactions without any meaningful dependence on the specific peptide sequence. The starting point of the reaction, the very reactive specie that undergoes these fatal structural changes, has not been identified yet. For a long while the conventional view has been that amyloid connected pathologies are brought about by the amyloid fibrils found in the plaques, but more recently it has been suggested that the main neurotoxic


Table 1.1: Diseases featuring amyloids.

Disease Protein featured Official abbreviation

Altzheimer’s disease Beta amyloid Aβ

Diabetes mellitus type 2 IAPP (Amylin) AIAPP

Parkinson’s disease α-synuclein -

Bovine spongiform PrP APrP


Huntington’s Disease Huntingtin -

Medullary carcinoma Calcitonin -

of the tyroid ACal

Cardiac arrhythmias Atrial natriureic factor AANF

Atheroscerosis Apoliprotein AI AApoA1

Reumatoid arthritis Serum Amyloid A AA

Prolactinomas Prolactin APro

Familial amyloid Transthyretin ATTR


Hereditary Lysozyme ALys

systemic amyloidosis

Dialysis rel amyloidosis β-2 microtubulin Aβ2M

Finnish amyloidosis Gelsolin AGel

Lattice corneal dystrophy Keratoepithelin AKer

Cerebral amyloid β amyloid Aβ

angiopathy Cystatind ACys

(icelandic type)

Systemic AL amyloidosis Immunoglobulin AL

light chain

Injection amyloidosis Insulin -

species would be the soluble oligomeric species. This suggests a protective role of amyloid fibrils, that could be part of a defence mechanism carried


Figure 1.5: (a)Schematic representation of fibrillation reaction. The reaction comprises three main steps. After a lag phase during which structural changes occur and nuclei form, the fiber starts elongating in protofilaments that eventually pack together to form a mature fibril.(b)Sigmoidal curve describing fibril increase during time. (c) Amyloid deposits found during the stationary phase. Picture from Ref. [24]

out by nature for misfolded intermediates quenching. Although in the last years several hints came out from experimental evidences of the misfolded intermediate the subject is still very debated and not yet clearly understood.

The irreversible nature of fibrillation, together with the difficulty in obtaining well diffracting crystals of fibrils have made very difficult the study of these misfolded states. After a lag phase, during which several nuclei (probably misfolded oligomers) form, the structure starts to polymerize through the stacking of the preformed nuclei. Fibrillation is a highly cooperative process and since heterogeneous nucleation mechanisms can take place, the presence of already formed amyloid fibrils is also able to catalyse the formation of new fibrils[25][26].


Figure 1.6: Schematic representation of all the possible pathways for a protein to undergo fibrillation. According to the most supported idea the amyloid-prone protein undergoes conformational changes following few main steps: partial un- folding, nucleation (during which a lag phase occur s), elongation and further aggregation of elongated structures. The seeds induced fibrillation is also reported in the picture. Picture from Ref. [15].

Amyloids Structure

Amyloid structure is built up by the stacking of repeated substructures, consisting of β-strands that run perpendicular to the fiber axis, forming a cross-β sheet of indefinite length[16].

It is thus made of an ordered arrangement of many (usually thousands) copies of the same protein. Amyloids are easily identified using electron mi- croscopy (EM) as long, unbranched filaments with diameters of 6-12 nm[36].

By definition, all amyloid fibrils have a translational symmetry element that lies parallel to the fibril axis, whereas electron micrographs indicate that most have in addition a rotational element (combined rotational-translational)


Figure 1.7: Extended parallel β-sheet structures deduced from solid state NMR studies. Picture from Ref. [16].

yielding a helical or screw symmetry. Thus, well-aligned fibrils can some- times give diffraction patterns that can be used to model (at low resolution) the fibril structure. Despite their highly ordered nature, amyloids are dif- ficult to study by high-resolution structural methods. The one-dimensional nature of the order in the fibrils makes them poor candidates for three- dimensional crystallization, and to date, the only representative crystal struc- tures are of amyloidogenic peptides that are short enough to pack into a three-dimensional lattice[35]. These structures indicate that the most basic cross-β structure is in fact a one dimensional crystal with single rotational and translational symmetry elements. Alternatively, solid-state nuclear mag- netic resonance (NMR) is a well-suited high-resolution structure method for the study of amyloids, yet to date, only a single high-resolution NMR struc- ture of an amyloid is available[17]. Whether the β-sheets composing the fibril structure are in parallel or antiparallelel configuration is still matter of de- bate, as also the eventual presence of residual folded structures in the fiber.

Experimental evidences have shown that, even if a common motif builds up the elongated structure, the structure characteristics of the stacked ele- ments, intermediate of the reaction, can be very different. FT-IR data have shown that, although the prevalence of intermolecular β-sheets is conserved in fibrils, different amyloidogenic proteins seem to assemble in different ways, arranging their skeletal β-sheets in parallel or antiparallel form[40][41]. More- over, even the same protein in different environmental or thermodynamic conditions may aggregate into distinct amyloids with their own secondary


structure peculiarities. In the absence of high-resolution fibril structures, increasingly credible models are being derived by integrating data from a cross fire of experimental techniques. A number of low resolution studies, aimed at the assignment of amyloids secondary structure (FT-IR, Circular Dichroism, and Raman spectroscopy) have reported the presence of folded regions in amyloids. Even if several works remarked the existence of par- tially folded segments in amyloid structure, the theoretical models proposed predict a full β-sheet composition[37] for most of known amyloids. Among the possible conformations proposed for amyloids, the ”strand-loop-strand”

motif has been so far the most supported. In this conformation, that is also found in globular proteins β, the two β-strands composing the fibrils interact one to each other through H-bonds between adjacent antiparallel β-sheets. Conversely, most current models of disease-related amyloids in- voke ”β-arcades”, columnar structures produced by in-register stacking of

”β-arches”. A β-arch is a strand-turn-strand motif in which the to β-strands interact via their side chains, not via the polypeptide backbone, as in a con- ventional β-hairpin. These two configurations are reported in Figure 1.8.

All models proposed, aimed at describing the structure of amyloids, can be divided into three groups:

1- Refolding models

2- Natively unfolded models 3- Gain of interactions models

Refolding models predict the partial unfolding of the protein prior to aggre- gation and have been suggested to be built up by parallel β-helices, although this specific structure would not seem to be a requirement of the concept of a refolding model (these model well described insulin fibrils structure). The natively unfolded models concern, conversely, all that proteins that are poorly folded (as for instance α-synuclein). Among these natively unfolded proteins, there are several that form amyloid like fibrils. In the process form- ing a fibril, all or part of the previously unstructured polypeptide becomes structured to form the cross β-spine. In the gain of interaction models


Figure 1.8: Cross-β structures can form in two ways. The first class(a) has two adjacent strands with a β-turn between them that form an antiparallel β-sheet called a β-hairpin. Two such folded chains form intramolecular H-bonds and their side chains interact with the other antiparallel β-sheet. In the second class(b), the polypeptide chain also folds back on itself, but the connected β-strands make contact via their side chains rather than interacting via H-bonds of the backbone.

As a result, the two linked strands, find themselves in two different parallel β-sheets with the other strand. This structure is called β-arcade.

a conformational change in a limited region of the native protein occurs, ex- posing a previously inaccessible surface. This newly exposed surface binds to a surface of another molecule, binding up the fibril. All the described models can be found in Figure 1.9. Up to now, what the real three-dimensional struc- ture of amyloids is, is still unknown and the several experimental evidences lead to the idea of a polymorphic nature of amyloids.


Figure 1.9: Models of amyloid structure. (a) Cartoon depicting the three gen- eral types of models for the conversion of proteins from their native state to the amyloid-like state. In refolding models, the protein unfolds and then refolds into a different structure, which is stabilized largely by backbone hydrogen bonds. In natively disordered models, the cross-β spine forms from protein segments that are poorly structured in the native state. (b) Ribbon diagram showing an example of a left-handed parallel β-helix, taken from the structure of UDP N-acetylglucosamine O-acyltransferase from Escherichia coli (PDB code 1LXA). This helix was used in modelling the structure of the refolded portion of prion protein in fibrils[47].

(c) Cartoon depicting the parallel superpleated β-structure proposed for Ure2p fibrils[46]. The view looks down the long fibril axis. Each arrow represents a view down a single β-sheet. The blue ovals represent the natively folded C-terminal domains, showing how stacked domains could pack around the serpentine core.

Picture from Ref. [37]


Figure 1.10: Gain of interaction models. (a) Cartoon depicting the four subtypes of gain-of-interaction models. In direct stacking models (panel i), the gained in- teraction is achieved via simple stacking of subunits. Alternatively, in the cross-β spine models (panel ii), a segment of the protein separates from the core domain to stack into a cross-β spine, with the core domain decorating the edges of the spine. In the somewhat more elaborate model shown in panel iv, the molecules at the edges of the spine domain swap with identical molecules. This permits a wider range of stable geometries around the cross-β spine. In the remaining subtype (panel iii), proteins first domain swap and then stack into the fibril. Picture from Ref [38]


1.2 High pressure bioscience

The realm of environmental extremes, that is the greatest portion of our biosphere, is unexpectedly densely crowded with barophilic and thermophilic (pressure and heat adapted) species. Psychrophilic barophilic (cold- and pressure adapted) species, which live at 2C are found in the deepest ocean floor (∼11000m) in the Mariana Trench and in deep sea sediments. Organ- isms far more complex than bacteria can be found generally several thousand metres under the sea surface in conditions of high hydrostatic pressure(∼300 bar) and high temperature (up to 120C). Those conditions that are com- monly considered extreme are actually bursting with forms of life that, quite amazingly, are not so different from those found on the surface of the earth.

Historically, the application of high pressure techniques to biological mat- ter has been most successfully carried out on food processing but in recent years[39] the interest in pressure as a thermodynamic variable has been grow- ing in physico-chemical studies of biological materials. Observations of the effect of high pressure on biological materials and organisms can be traced back to the last century. High pressure treatment to kill bacteria was first described in 1895 by Royer[18]. Hite and coworkers[19] of the University of West Virginia reported the use of high pressure for the preservation of milk in 1899. Bridgman[20] observed coagulation of egg white by high pres- sure treatment in 1914. Since these reports, the research on the effect of high pressure on biological materials and living organisms have continued without interruption. However, it is surprising that attempts to apply high pressure to food science almost stopped for 70 years since the pioneering at- tempts of Hite and co-workers until recent break-throughs in Japan, where high pressure-processed foods have been successfully released on the food markets. In principle, high pressure inactivates micro-organisms, denatures proteins, and gelatinizes starches. These properties of high pressure are sim- ilar to the effects given by temperature. However, unlike temperature, high pressure keeps natural flavour, color and nutrients of natural foods, in other words, preserves the original properties of the biological material. Therefore,


high pressure treatment may be applied not only to food materials but also to biological matter such as organs and tissues. It is natural that high pres- sure technology is extended to bioscience and biotechnology in general and demand for this extension are increasing under the support of successful use of high pressure in food science and technology.

1.2.1 High Pressure Principles

High pressure is able to perturb biosystems in a way that can be con- sidered unique. The perturbation induced can produce the most disparate variety of effects, in a way that is often reversible thus enlightening funda- mental aspects that are otherwise hidden at ambient pressure. To describe high pressure effects it can be useful to explicitate pressure and temperature dependence of Gibbs Free energy function, reported in Equation 1.1.

d(∆G) = ∆V dp − ∆SdT (1.2)

While a temperature change, that is connected to entropic variations, in a biochemical system produces a simultaneous change in thermal energy and volume, by carrying out high pressure experiments at a constant temperature one can tune the energetic of the system affecting only the volume-dependent part of the energy, thus enabling the separation of thermal and volume ef- fects. Moreover, if we consider the typical volume variations encountered in biological processes we found that , in contrast to the phase transitions occurring in molecular systems, covalent bond angles and bond lengths can be considered to remain unchanged in the pressure range that is relevant to biological systems. For instance, a protein unfolding process is accompanied by a volume change of about -30 ml mol−1 and occurs at a pressure of about 5 kbar. The total energy input in the system is only of about 15 kJ mol−1. Consequently, pressure enables one to tune the non-covalent interactions, and in particular the hydrophobic effect and hydrogen bonding, that are respon- sible for the stabilization of biological molecules. High pressure also affects chemical equilibria and reaction rates. Le Chatelier’s principle, that can be


deduced from Equation 1.3 governs the behaviour of all systems under pres- sure. It predicts that the application of pressure shifts the equilibrium of a reaction towards the state that occupies a smaller volume, and accelerates such processes for which the transition state has a smaller volume than the ground state.




= −∆V

RT (1.3)

Here k is the reaction rate constant, P is the pressure, R is the gas constant and T is the temperature. The activation volume ∆V is the difference in partial molar volume between the transition state of the reaction and the reactants. By comparing the reaction rates at different pressures it is so possible to draw valuable conclusions about the nature of the reaction and its mechanism. Often, due to the complexity and the variety of interactions involved, the effect of pressure on biosystems cannot be predicted, and high pressure experiments lead to the discovery of new phases and processes not accessible at ambient pressure.

1.2.2 High Pressure stability of Proteins: elastic and plastic effects

For a protein in acqueous solution, the partial molar volume Vprotein com- prises contributions from three terms[22], respectively:

Vprotein = Vatoms+ Vvoids+ ∆Vhydration (1.4) where Vatoms is the sum of the volumes of the constituent atoms of the pro- tein, Vvoids is the volume of void space within the protein, and ∆Vhydrationis a system volume change that results from protein-solvent interactions. Vatoms is a fixed quantity for a given protein. Thus, all the reactions triggered by pressure lead to a variation in the other two contribution factors. This means that high pressure conformational changes induced on proteins, mainly af- fect the hydration state of the protein and its void volume, thus its tertiary


and quaternary structure. By integrating Equation 1.2, we find the expres- sion for the free energy change during protein denaturation (for a two-state transition):

∆G = ∆β

2 (P − P0)2 + ∆α(P − P0)(T − T0)




T0 − 1

 + T0

+ ∆V0(P − P0)

−∆S0(T − T0) + ∆G0


where ∆G is the free energy change of denaturation, ∆α = (∂∆V /∂T )P,

∆β = (∂∆V /∂P )T , Cp is the heat capacity, and the subscript 0 refers to a reference temperature and pressure. This equation predicts that there is a close stability(∆ G = 0) contour in P-T space, as shown in Figure 1.11 Thus, processes resulting in overall decrease in system volume are favoured at

Figure 1.11: Schematic representation of pressure-temperature stability contour of proteins. within the contour the native state is stable. Note that the model predicts two denaturation temperatures at ambient pressure.

high pressures, whereas those that result in volume increase are disfavoured.

For the specific case of protein structural transitions, three processes de- serve particular attention. First dissociation of chemical species into their respective ions contributes to the overall reduction in system volume due to electrostriction[21]. Thus any transition that results in deprotonation to form


charged residues is favoured at high pressures. Both the reaction reported in equations 1.6 and 1.7, as reported by Van Eldik et al [23], are associated by a negative volume variation of the system.

protein − COOH → protein − COO+ H+ (1.6)

protein − N H2+ H2O → protein − N H3++ OH (1.7) this means that, structural transitions with concomitant formation of charged residues in general should be favoured at high pressure. Thus, salt bridges may be disrupted and the pressure mediated increases in protein charge will serve to increase electrostatic repulsion between protein molecules, thus de- creasing the rate of reaggregation. Moreover the exposure of hydrophobic groups to water is also favoured under high pressure. In contrast, the change in volume for the formation of a hydrogen bond is associated with a very small partial volume change. This means that high pressure only have little effects on the tendency of a protein to form hydrogen bonds. As reported in Figure 1.11, two stability regions can be drawn for proteins on pressure- temperature plane. Around ambient conditions, when the protein is in its na- tive state, a variation in thermodynamic parameters results in elastic changes (reversible)[34]. Conversely, at high pressure and high or low temperature, changes are plastic or conformational (in most case irreversible). In the elas- tic region the secondary structure of the protein is preserved, while changes in the bond length, hydration and cavities that result from the imperfect packing in the protein interior may be expected. If the temperature or the pressure is high enough, also cooperative changes in the secondary structure may result, leading to plastic or conformational changes.

High pressure crystallization

Since hydrostatic pressure affects the whole system uniformly, it is ex- pected to be an important parameter to control protein crystallization. A number of studies on high pressure crystallization of proteins have been


reported[43][44]. High pressure can drive crystallization, affecting both crys- tal growth rate and solvent viscosity. The effect of pressure in increasing of decreasing crystallization rate, is strictly protein dependent. For in- stance, the crystal growth rate of glucose isomerase increases with increasing pressure[45], while that of tetragonal lysozime crystal[43] decreases. The pressure usually employed in high pressure crystallization, range up to 1-2 kbar.

High pressure unfolding

As discussed in section 1.2.2, there exists a close P-T region in the Gibbs free energy profile in which the native protein state is favoured. According to high pressure principles, increasing pressure, we expect to unfold the protein acting mainly on weak interactions, without directly affecting its H-bonds state. This means that, quaternary structure breakage and tertiary struc- ture opening will occur and eventually, the destabilization of this structures could lead to the cooperative opening of the secondary structure. Theoreti- cal models have predicted that pressure unfolding mechanism occurs through the weakening of van der Waals, electrostatic and hydrophobic interaction due to water penetration inside the protein hydrophobic core, with the con- sequent exposure of the hidden surface to water (see Figure 1.12). The high

Figure 1.12: Proposed mechanism of pressure unfolding of proteins from cite.


pressure unfolded state might be, thus, very different compared to the heat induced one. The high pressure unfolding of proteins can sometimes lead to a protein state that becomes aggregation-prone if pressure is quickly decreased to ambient[42].

Misfolding at high pressure

As described in section 1.2.1, high pressure shifts the system towards the state that occupies a smaller volume. Thus, high pressure is expected to disaggregate aggregated assemblies of proteins. Moreover, due to the high cooperativity proper of the aggregation of misfolded proteins, the pressure induced opening of the secondary structure is also expected due to the loss of stability of aggregates, whose structure is thought to be driven by elec- trostatic and hydrophobic interactions. The changes that can be observed subjecting and aggregate specie to high pressure range for this reason in conformational (plastic) region of P-T stability diagram. The application of high pressure on amyloids can be of fundamental importance for two main reason. First, whether electrostatic interactions and hydrophobic effect or H-bonds are the real stabilizing forces of amyloid structure is under debate and due to its very selective action, high pressure offers a very important tool for a deeper understanding of this point. Moreover, due to its little effect on H-bonds stabilized structures, it can induce the stabilization of on pathway intermediates not detectable at ambient pressure due to the high cooperativity of fibrillation.



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Chapter 2


In this chapter Fourier Transform Infrared spectroscopy (FTIR) princi- ples and their application to the study of proteins will be discussed. Moreover, we will describe the apparatus that has been used to perform FTIR measure- ments. Then, the high-pressure techniques will be introduced together with synchrotron radiation and the experimental station of SISSI infrared beam- line at ELETTRA Sincrotrone Trieste.

2.1 Infrared Spectroscopy

Infrared(IR) spectroscopy is one of the oldest and well established exper- imental techniques for the analysis of secondary structures of polypeptides and proteins[1]. Fourier Transform Infrared Spectroscopy (FT-IR) is recog- nized as a valuable tool for the examination of protein conformation in H2O- based solution, as well as in deuterated forms and dried states, resulting in a greatly expanded use in studies of protein secondary structure and pro- tein dynamics in the past decade. Although X-ray crystallography provides the most detailed information concerning positions of individual atoms in the protein structure, it is not, however, possible for all proteins to form a quality crystal for such analysis. In addition, the crystallographic data of a protein cannot be easily extrapolated to the dynamic properties of the proteins in



solutions. Nuclear magnetic resonance spectroscopy can be an alternative to X-ray crystallography in solution, but the interpretation of nuclear mag- netic resonance spectra of a large protein is a very cumbersome process[2].

Thus, the low resolution techniques, such as FT-IR and circular dichroism (CD), are important and commonly used techniques for protein structure and dynamics studies. FT-IR spectroscopy, in particular, has proven to be very useful in the study of amyloid fibrils, proteinaceous aggregates linked to the development of several important diseases (see Chapter 1). Due to the difficulty in obtaining well diffracting amyloid crystals, it has not be possible to solve their structure at atomic resolution so that, all we know about amy- loids morphology and structure comes from low resolution techniques. The spectral range covered by FT-IR goes from far infrared, around 1 meV, up to UV light, around 5 eV. The non-invasive nature of the infrared radiation, the short time required for a measurement (due to Fourier Transform implemen- tation) and the versatility of the infrared experimental set-up (that enable the coupling of FTIR with other techniques, as for instance High Pressure techniques), make FT-IR the best low-resolution technique for the evaluation of protein structure and for the study of unfolding and misfolding kinetics.

2.1.1 FT-IR principles

In infrared absorption theory covalently bonded atoms are treated as har- monic oscillators. For non-interacting molecules consisting of N atoms there are 3N-6 vibrational degrees of freedom (3N-5 for a linear molecule), which is equivalent to state that all vibrational distortions can be described as the sum of 3N-6 ”fundamental” vibrational modes. According to a semi-classical approach, each of the fundamental vibrational modes will have a frequency determined by the forces exerted on the atoms as they move from the equi- librium molecular configuration in the manner required by that mode. For vibrational spectroscopy, electric dipole interactions with the electric field are totally dominant, and the interactions with the magnetic field can be ne- glected. The quantum-theoretical analysis shows that a necessary condition


for the electromagnetic radiation to interact with a material passing through it, is the match of its frequency to that of a fundamental mode of the molecule studied. Moreover it states that there must be a change in dipole moment before absorption of the radiation may occur. If we consider a diatomic molecule, a necessary condition for the occurrence of infrared absorption is the presence of a permanent dipole moment on it. During molecular vibra- tions the displacements of the atoms from their equilibrium positions are so small that the forces on the nuclei can be considered linearly proportional to the displacement x (Hooke’s law), and for displacements from equilibrium these must be towards the rest position (i.e. negative). In the simple case of the diatomic molecule vibration, we can write:

F = −kx = −~ ∂2V

∂x2 = µ∂2x

∂t2 (2.1)

where µ is the reduced mass of the system µ1µ21+ µ2, V is the energy potential and x is the displacement form equilibrium. The equation 2.1 describes the harmonic oscillator problem and its solution is:

ν = 1 2π


µ (2.2)

The equation 2.2 connects the infrared absorption of the molecule to structural parameters, as bond energy, through the elastic constant k. Ob- viously, in the case of N-atomic molecules the problem is much more com- plicated and its solution is not trivial. Fourier Transform IR is usually per- formed with spectrometers (based on interferometry). A simple scheme of a Michelson interferometer is reported in Figure 2.1.

Therein, a radiation beam produced by a source(S) is split in two com- ponents that perform two different optical paths. When those components are combined again, the resulting electric field is an oscillating function of the optical path difference δ, because of interference. The recombined in- tensity I(δ), called interferogram, is in the simple case of monochromatic electromagnetic radiation a periodic sine function. When instead broadband radiation illuminates a Michelson interferometer I(δ) results in a combina-


Figure 2.1: (left)Schematic representation of Michelson interferome- ter.(right)(up) source signal in the time domain collected at the end of the in- terferometer (interferogram).(down)Source spectrum in the frequency domain.

tion of all the components of the spectrum, each multiplied by its intensity.

Therefore the interferogram implicitly contains information over the whole frequency dependence of light and its Fourier transform I’(ω) is the power spectrum of the radiation. A formal description of principles of FT-IR can be found in Ref.[3, 4]. Through the use of modern interferometers it is, more- over, possible to perform measurements with a high resolving power (that is proportional to δ’s maximum value, i.e. the length of the arm of the inter- ferometer) and using circular apertures instead of slits as in monochromator (Jaquinot advantage). All frequencies are measured simultaneously, thus re- ducing the collecting time by a factor of n (where n is the number of points in the spectrum) and improving the signal to noise ratio by a factor of N2 when acquiring and averaging N interferograms with respect to the same spectrum acquired with other techniques (Fellget advantage). The position of the moving mirror is determined by measuring the fringes produced by a monochromatic laser source that passes through the same optical path of the radiation. This ensures that the value of δ is determined with ”optical”


precision (Connes advantage). The laser beam also can be used to check the parallelism of the fixed and moving mirrors with respect to the radiation beam.

2.1.2 The Absorbance

FT-IR spectroscopy on proteins is usually employed though the measure- ment of the absorption wavelength and intensity of a proteic sample in the mid infrared range. All the experiments reported in this thesis are based on absorption measurements, giving rise to ”Absorbance” spectra. Absorption in the IR region arises mainly from excitation of molecular vibrations. The absorbed signal is evaluated by sending the modulated light, coming out from the interferometer, through the sample and measuring the transmitted inten- sity. Lambert Beer law, relates the absorption of light to the properties of the material through which the light is travelling stating that the transmitted light can be expressed by the equation:

I = I0eαx (2.3)

Where I0 and I are respectively incident and transmitted light, α is the absorption coefficient proper of the medium crossed and x is the distance travelled by light through the medium (optical path-length). The absorption coefficient, that depends on the number of absorbers per unit of volume and on how strongly a chemical species absorbs can be written as:

α = N  (2.4)

Where N is the number of absorbers per unit of volume and  is the molar ab- sorbitivity (or extinction coefficient), that takes into account the absorption efficiency. To obtain a quantity proportional to the number of absorbers, it is useful to consider the linear form of Lambert-Beer law, and to introduce the Absorbance, defined as:

A = −LogI

I0 = αx = Cx (2.5)


Where C, for liquids, is the concentration of absorbers. Infrared absorp- tion spectroscopy has been widely used in biology and biophysics due to the many absorptions that originate, in particular in the region between 1000 and 4000 cm−1, by biological molecules. In Figure 2.2 it is reported a typical

Figure 2.2: Typical mid infrared spectrum of a cell from Ref.[5].

spectrum of a cell(from Ref.[5]). It is easy to recognize, from the observa- tion of the spectrum of a cell, the contribution of lipids, proteins and nucleic acids.

2.1.3 FTIR on proteins

FT-IR evaluation of the secondary structure of Proteins

The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat unit[9, 10, 11]. The polypeptide and protein repeat units give rise to nine characteristic IR absorption bands, namely, amide A, B, and I-VII. Among these, the amide I and II bands are the two most prominent vibrational bands of the protein backbone[13, 14, 15].

The amide I mode is primarily a C=O stretch band (approximately 80%), with some contributions from C-N stretch and C-C-N deformation (see left


panel in Figure 2.3). The amide II mode is an out of phase combination of

Figure 2.3: Schematic representation of amide I and amide II vibrational ab- sorptions of proteins found in the mid infrared range.

largely N-H in plane bend (40-60% of the potential energy) and C-N stretch (18-40%) and shows much less protein conformational sensitivity than the amide I contribution (see right panel in Figure 2.3). The C=O stretch is in fact affected by the H-bond state of oxygen, so that different secondary structure arrangements give rise to distinct absorption peaks, shifted of few cm−1 one to respect to the other. The amide I band is thus composed of several overlapping peaks, that reflect the precise secondary structure com- position of the protein. There exists a huge database in which each amide I frequency is connected to a different secondary structure. These assignments have been made from model proteins of known structure (usually from X-ray crystallographic data).

Amide I deconvolution

The Amide I deconvolution is a very reliable technique and so far, it has been widely used in the evaluation of the secondary structure of proteins.

The absorption ranges of all the amidic vibrational modes are reported in Figure 2.4 and a more detailed discussion of their origin can be found in Ref.


[16, 17].

Figure 2.4: Amide I/I’(deuterated form) frequencies for protein secondary struc- tures. Amide I(in H2O):dashed line frames. Amide I’(in D2O): solid line frames.

α: α-helices. In highly solvent exposed α-helices, the amide I’ band can shift to 1630-1645 cm−1due to additional hydrogen bonding of the solvent accessible C=O groups to water. The helical band in membrane proteins typically occurs between 1657 and 1662 cm−1which has been suggested as indicative for more flexible and/or more stretched α-helices.β:β-sheet structures. For proteins often more than one component is observed. This reflects differences in hydrogen bonding (the stronger and shorter the hydrogen bond, the lower the frequency) as well as differences in transition dipole coupling in different β-strands. β#: β-strands in aggregated structures. IR spectra of thermally aggregated proteins are characterized by a sig- nificant increase in splitting between the low- and high-frequency β-components (β and β respectively) in comparison to that observed from β-sheets in native proteins, indicating intermolecular β-sheet structure with very strong hydrogen bonds. Similar features are found in IR spectra of amorphous protein deposits (as in inclusion bodies) or ordered protein aggregates (as in amyloids), U: unordered, lp:loops, t:turns.

In Figure 2.5 is reported an example of deconvolution of the Amide I


band. The standard deconvolution technique is based on three main proce- dures. In the first step, the presence of bands arising from amino acid side chains must be recognized before attempting to extract structural informa- tion from the shapes of amide I band. The contribution of the side chain vibrations in the region between 1710 and 1580 cm-1 (amide I region) has been thoroughly investigated by Venyaminov and Kalnin[18]. Among the 20 proteinogenous amino acids only 9 (Asp, Asn, Glu, Gln, Lys, Arg, Tyr, Phe, His) show a significant absorbance in the region of interest for secondary structure evaluation[19]. A visual recognition of the main overlapped back- bone absorptions can be done by the observation of the second derivative spectrum of absorbance. It is in fact possible to assign the position of the overlapped peaks, to the minima of the second derivative spectrum. The sec- ond derivative analysis enable the assignment of the number of overlapping peaks and of their energies but can not be considered a quantitative deconvo- lution method. From quantum theory we know that, due to the finite lifetime of excited levels, the absorptions undergo a Lorentzian spreading. Additional disorder effects (doppler broadening) can lead to a further spreading of ab- sorption peaks, that is, to its own nature, well described as a Gaussian effect.

For infrared absorptions in liquids it is almost always necessary to consider the combination of the two effects. For this reason, the deconvolution proce- dure is performed modelling the absorptions with a function resulting from the convolution of the two spreading mechanisms (Voigt profiles). While the amide I band is only slightly sensitive to deuteration, the isotopic exchange can shift the amide II band towards lower frequencies of about 100 cm−1. The amide II band is in fact mainly composed of NH vibrational modes, occurring at about 1550 cm−1 that turn into ND modes, occurring at about 1450 cm−1 if exposed to deuterated environment. The amide II sensitivity to H-D exchange enable us to follow the opening of the tertiary structure of the protein monitoring the consequent exposure of buried residues to the solvent through the deuteration of their backbone segments.


Figure 2.5: Amide I deconvolution procedure.(up) Experimental and fitted data, obtained from the deconvolution of the Amide I band in 5 Voigt profiles.(down) 2nd-derivative spectrum of Amide I band. It is possible to recognize 5 minima, assigned to the 5 components that have been used in the deconvolution procedure.

The problem of water absorption

One of the most important advantages offered by FT-IR spectroscopy over other techniques, is the possibility to study biomolecules in physiolog- ical condition. The presence of water absorption can make the study of biological molecules hard, and imposes the necessity to develop strategies to subtract its contribution from the sample spectra. In Figure 2.6 are reported the spectra of pure water and deuterated water in the mid infrared range.

As we can see from the observation of Figure 2.2 and 2.6, there are several superposition of absorptions coming from biomolecules. In particular the most interesting absorptions coming from proteins, that enable the evalua- tion of their secondary structure, fall in the region around 1650 cm−1[6]. As


Figure 2.6: Absorbance of proteins in H2O and D2O, in the spectral range between 4000 and 1000 cm−1.

we can see from the observation of Figure 2.6, the spectrum in this region is clearly dominated by water absorptions. To overstep this problem, two different strategies are widely used, according to the characteristics of the


specific sample under inspection. A first approach, mainly used on complex structures, as for instance cells, that has been widely and successfully applied to proteins as well consists in the subtraction of water contribution. Many algorithms have been proposed to best subtract background water. All of them try to find the exact percentage of water inside the sample. They are implemented through the comparison between sample solution and pure wa- ter spectra to find the real water content inside the protein solution. Thus, a linear subtraction normalized to the water percentage found by the spec- tra comparison is performed[7]. This strategy imposes the necessity to have not saturated bands around the spectral region of interest. For this reason, the limitation of the optical path to 6 µm is required. Another strategy, widely used in the study of proteins, is the substitution of water with heavy water(D2O). The isotopic exchange leads, in fact, to the shift of the vibra- tional modes of water towards lower frequencies (see Figure 2.6), without affecting proteins conformations[8]. This approach enables the use of optical path-lengths one order of magnitude wider (∼50 µm). Moreover, the isotopic exchange influences important proteins bands, giving important informations about their solvent exposed surface(this point will be discussed in the next section).

2.1.4 High Pressure effects on proteins vibrational modes

As described in the previous section, FT-IR represents one of the most challenging techniques in the evaluation of the secondary structure of pro- teins. For this reason it is particularly suitable for the study of misfolding, a process during which the protein completely looses its secondary struc- ture and rearranges itself into a β-sheet conformation. The addition of a high pressure perturbation to the FT-IR spectroscopic characterization of proteins widely enhances the sensitivity of FT-IR spectroscopy, allowing to probe the conformation of a protein from the high pressure behaviour of its vibrational modes.


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