Prırodovedecka fakulta Univerzity Karlovy v Praze

Katedra fyzikalnı a makromolekularnı chemie

Charles University in Prague, Faculty of ScienceDepartment of Physical and Macromolecular Chemistry

Modelovanı chemickych vlastnostı nano- a biostrukturModelling of Chemical Properties of Nano- and Biostructures

Autoreferat disertacnı praceSummary of the Ph.D. Thesis

Interakce iontu s proteiny

Ion-Protein Interactions

Mgr. et Mgr. Jan Heyda

Ustav organicke chemie a biochemie, AV CR v.v.i.Centrum biomolekul a komplexnıch molekulovych systemu

Skolitel/Advisor: Prof. Pavel Jungwirth, DSc.

Praha, 2011

CONTENTS 2

Contents

1 Abstract in English 51.1 Introduction into complex systems . . . . . . . . . . . . . . . . . 51.2 Aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.3 Computational methods . . . . . . . . . . . . . . . . . . . . . . . 6

1.3.1 Simulation protocol . . . . . . . . . . . . . . . . . . . . . . 61.3.2 Methods of analysis . . . . . . . . . . . . . . . . . . . . . . 7

1.4 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . 81.4.1 Case study – Model of the peptide bond . . . . . . . . . . 91.4.2 Amino acid proxies . . . . . . . . . . . . . . . . . . . . . . 101.4.3 Single amino acids and short oligopeptides . . . . . . . . . 101.4.4 Peptides and proteins . . . . . . . . . . . . . . . . . . . . . 11

1.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2 Autoreferat v ceskem jazyce 132.1 Uvod do komplexnıch systemu . . . . . . . . . . . . . . . . . . . . 132.2 Cıle prace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.3 Vypocetnı metody . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.3.1 Simulacnı protokol . . . . . . . . . . . . . . . . . . . . . . 142.3.2 Druhy analyzy . . . . . . . . . . . . . . . . . . . . . . . . 15

2.4 Vysledky a diskuse . . . . . . . . . . . . . . . . . . . . . . . . . . 152.4.1 Prıklad – studie modelu peptidove vazby . . . . . . . . . . 162.4.2 Modely funkcnıch skupin . . . . . . . . . . . . . . . . . . . 182.4.3 Jednotlive aminokyseliny a kratke oligopeptidy . . . . . . 182.4.4 Peptidy a proteiny . . . . . . . . . . . . . . . . . . . . . . 19

2.5 Zaver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Bibliography 21

Curriculum vitae 24

Selected Publications/Seznam publikacı 25

Title: Ion-Protein InteractionsAuthor: Mgr. et Mgr. Jan HeydaDepartment: Physical and Macromoleculer ChemistryAdvisor: Prof. Pavel Jungwirth, DSc., IOCB AS CR, v.v.i.Advisor’s e-mail address: pavel.jungwirth@uochb.cas.cz

Abstract: Conventional molecular dynamics simulations in combination withadvanced methods of analyses were used to improve the understanding of theinteraction between ions and proteins in salt solutions. Thus systems of diversecomplexity and size were investigated, starting with simple (and molecular) saltsolutions with small fragments that mimic the various functional groups of aminoacids such as N-methylacetamide representing the peptide bond or alkylated am-monium cations.

Continuing with individual positively charged amino acids (arginine, histidine,lysine) a strong binding interaction with small fluoride anion that is significantlyweakened for larger halides (Cl−, Br−, I−) was described. This observation wasextended by detecting the strong sensitivity of fluoride to charge distribution onammonium, lysine side chain, and the N-terminal of glycine while sensitivity ofiodide was found to be low. Later it was shown that the attractive side chain-sidechain interactions are significant for short positively charged peptide fragments inpolyarginine and dihistidine, while they are not present at all in case of polylysine.

Considering the qualitative difference in the origin of ion-specific interactions,electrophoretic mobility measurements (for mono- and tetra- amino acids) wereemployed in tandem with MD simulations. The ion-specific arginine-sulphate andarginine-guanidinium interactions were proved, both pronounced as the specificdecrease or increase in electrophoretic mobility in contrast to observations forlysine, chloride anion, and sodium cation.

Cation-specific interaction was found, both experimentally and computation-ally, to be responsible for specific affecting of the enzymatic activity of HIV-1protease and LinB enzyme from dehalogenase family. In both cases the generalsalting out effect was experimentally observed (pronounced as the increase of theenzymatic activity). Finally, the denaturant-specific unfolding pathway of Tr-pCage minipeptide was identified by comparing the denaturation process in ureaand guanidinium chloride solution.

In all the studies the aim was to shed more light on complex behaviour causedby ion-specific effects such as ordering in Hofmeister series, speeding up the en-zymatic activity, preferential interactions with functional groups or the osmolytespecific denaturation pathways.Keywords: molecular dynamics, proteins, denaturation, salts, osmolytes.

Nazev prace: Interakce iontu s proteinyAutor: Mgr. et Mgr. Jan HeydaKatedra: Fyzikalnı a makromolekularnı chemieVedoucı doktorske prace: Prof. Pavel Jungwirth, DSc., UOCHB AV CR, v.v.i.E-mail vedoucıho: pavel.jungwirth@uochb.cas.cz

Abstrakt: V predkladane praci byly pouzity metody molekulove dynamiky vkombinaci s pokrocilymi technikami analyzy k zıskanı detailnıch informacı apro hlubsı pochopenı interakcı mezi ionty a proteiny v roztocıch. Proto bylyzkoumany systemy o ruznem stupni komplexity, pocınaje roztoky molekularnıchsolı s drobnymi fragmenty, podobajıcımi se funkcnım skupinam aminokyselin,jako napr. N-methylacetamid reprezentujıcı peptidovou vazbu nebo alkylovaneamonne kationty.

Dale se predmetem naseho studia staly jednotlive kladne nabite aminoky-seliny, u nichz byla popsana silna interakce s malym fluoridovym aniontem, jez jevsak pro vetsı halogenidy (Cl−, Br−, I−) vyrazne zeslabena. Toto pozorovanı byloprohloubeno objevem vysoke citlivosti fluoridu na rozlozenı naboje na amonneskupine, bocnım retezci lysinu a N-konci glycinu, zatımco jodid zde vykazovalpouze velice nızkou citlivost. Nasledne bylo prokazano, ze u kratkych kladnenabitych peptidovych useku v polyargininu a dihistidinu jsou preferovane pritaz-live interakce mezi bocnımi retezci, naopak v prıpade polylysinu prıtomny nejsou.

Na zaklade existence kvalitativnıho rozdılu v puvodu iontove specifickych in-terakcı bylo spolecne s MD simulacemi provedeno merenı elektroforetickych po-hyblivostı (pro mono- a tetra aminokyseliny). Tımto zpusobem byly odhalenyiontove specificke interakce mezi arigininem a sulfatem, a mezi argininem a gua-nidnym kationtem – oba efekty se projevily jako charakteristicke zvysenı ci snızenıelektroforeticke pohyblivosti ve srovnanı s lysinem, chloridovym aniontem a so-dıkovym kationtem.

Dale bylo experimentalne i vypocetne zjisteno, ze enzymaticka aktivita HIV-1 proteazy a enzymu LinB z rodiny dehalogenaz souvisı s kationtove specifickouinterakcı, ktera vysvetluje nektere zmeny v aktivite. V obou prıpadech byl expe-rimentalne pozorovan i efekt vysolovanı (v podobe zvysenı enzymaticke aktivi-ty). V neposlednı rade byly srovnany denaturacnı procesy probıhajıcı v roztocıchmocoviny a chloridu guanidneho a identifikovany dva ruzne zpusoby rozbalenıminipeptidu TrpCage.

Ve vsech zmınenych studiıch bylo snahou vıce osvetlit komplexnı chovanı vyseuvedenych systemu, a to zejmena objasnit iontove charakteristicke jevy, jako napr.poradı v Hofmeisterove rade iontu, urychlenı enzymaticke aktivity, favorizovaneinterakce nebo prubeh denaturace zavisly na danem osmolytu.Klıcova slova: molekulova dynamika, proteiny, denaturace, soli, osmolyty.

1 Abstract in English 5

1 Abstract in English

1.1 Introduction into complex systems

Biomolecules are always in contact with solvents and solvents are rarely ion-free. Proteins in salt solutions are examples of complex systems which are ar-guably the most popular research targets in contemporary computational sci-ence [1–4]. The number and quality of publications rise in hand with increasingand persuading evidence of which effects are robust and which are not. In simplesystems – such as binary or ternary solutions of electrolytes/non-electrolytes –it is becoming increasingly difficult to provide so needed novelty for high-qualitypublications.

However, recent achievements appeared even in the field of neat water. Thewater is ‘alive’ and challenging as documented by examples, such as Laage [5],who studied water reorientation and hydrogen-bond cleavage, Sedlmeier [6], whocritically compared water models with structure factors from neutron scatteringexperiment, or Dzubiella [7], who introduced a new generation of implicit solvents.

In any case once the complex species, either polymer, peptide or even a pairof large spheres, is introduced into the simple solution, the number of study op-portunities quickly increases [2,8–10]. Not only the general presence and evidenceof an effect, but rather its absolute strength is important. It is typically the de-tailed balance between competing interactions (i.e. electrostatics vs. solvation)that makes this research relevant for biological systems.

Proteins are very individual objects, therefore there are not many generallyvalid rules. However, the building blocks, twenty amino acids connected by thepeptide bonds, are always present. In the crudest approximation we can assign allthe relevancy to the functional groups, shown in Figure 1, and neglect everythingelse. This leads to a study of charged groups (carboxyl, ammonium, imidazolium,guanidinium), polar groups (alcohols, thiols, carboxamide), and also the peptidebond. It is not self evident if this is the correct approach.

A breakthrough appeared in 2007, when Auton, Holthauzen and Bolen [11] re-did the analysis of the old experimental data from the 1970s. Their study broughta completely new insight into interpretation of protein denaturation and reestab-

Figure 1. The functional groups that are supposed to be crucial for local inter-action of salts and solvent with proteins and peptides. A charged carboxyl groupof aspartate, glutamate and C-terminal (A), ammonium moiety of lysine or N-terminal (B), guanidinium moiety of arginine (C), imidazolium moiety of histidine(D), alcohols and thiols (E), carboxamide group of asparagine and glutamine (E),and the peptide bond (H).

1.2 Aims of the study 6

lished the validity of the so called transfer model [12], which is essentially nothingbut the reductionist approach of cutting into pieces. This brought a lot of enthu-siasm between theoreticians, as the simplified models are not stand-alone projectsany more.

1.2 Aims of the study

We would like to deal with the systems in their full complexity, but there isusually no other way to proceed further than to use approximations. Thereforethe reductionist approach is often used with advantage and a system is dividedinto pieces using the best accessible knowledge, or by a methodology that hasshown to be recently the most successful and promising.

The usual progress towards the understanding of ion-protein interactions wouldstart with ion-ion, ion-amino acid proxy, ion-amino acid, and ion-peptide com-plexes so as to naturally reach the ion-protein system. Even though the first twosteps capture most of the essence, the practical aspects (keeping the bio-track)force us to work in parallel on all fields.

We started with ion-protein studies [13, 14] from the very beginning, havingin mind that these systems with their wide range of interactions are a challengingtask. The effects of sodium and potassium cations were investigated, and follow-ing the protocol of Vrbka et al. [2], we quantified the sodium preference whencompared to potassium. However, the simulation data provided us with muchmore. The complexity encountered was another goal of these studies. In this waywe faced the upcoming obstacles and learned which effects need to be carefullytreated.

While the ion-protein projects took long time, putting in front of us newobstacles and challenges, both of the methodological and fundamental origin, itsupplied us continuously by new phenomena and effects that were itself of aninterest and many smaller projects consequently arose.

1.3 Computational methods

1.3.1 Simulation protocol

I focused on extensive direct classical molecular dynamics (MD) calculations,since my projects were quite suited for this technique. Also, in some of my studiesthe time-evolution was an important aspect [15], or even a key factor if the effectwas dynamic [14].

The system for the ‘data-harvesting’ part of the simulation can be usually pre-pared in the following way. The salt and water molecules are mixed in amounts soas to reach the desired concentration and so that the simple energy minimizationavoids the potential problems of close contacts. We are employing the periodicboundary conditions (PBC): an infinite number of periodic images of the originalsystem are placed in all three dimensions, giving rise to the effectively infinitesystem (this is the common, but vital trick). System is heated and pressurized tothe target temperature and density.

One can now insert (expose) any object into the solution, let the system evolve(propagate) in time, visiting relevant configurations and collect the data for the

1.3 Computational methods 7

purpose of the analysis. Some of the analyzing techniques are described in thefollowing section.

Note that the atoms inside the system move in time (the time step is usually1 fs) according to Newtonian equations of motion, interacting with each other viathe so called force-field (the effective empirical potential).

1.3.2 Methods of analysis

For basic analyses we used the internal module PTRAJ [16]. An importantexample is the so called radial distribution function (RDF), which expresses theprobability of finding two bodies in a given distance. It is the central object in thestatistical thermodynamics, directly related to thermodynamics. To understandthe ion-specific effects in different contexts we needed methods to be descrip-tive enough to capture both quantitative and qualitative pictures. These moreadvanced tools were coded as Python scripts. Two of them, descriptive by theirnature – the spatial distribution function and the proximal distribution functionare briefly described below.

• The spatial distribution function provides a description of environmentaround the central particle with three dimensional resolution. Thus for ageneral (non-spherical) object, it is significantly more descriptive than theconventional one dimensional radial distribution function. An example ofdistribution of sodium cation and chloride and iodide anions around N-methylacetamide is given in the left side of Figure 3.

• The proximal distribution function is one dimensional, but unlike RDF(implicitly assuming sphericity) it takes into account the real shape of thecentral molecule, therefore is suited for descriptions in complex systems.Division of N-methylacetamide in polar peptide bond and ‘hydrophobicmethyl groups’ is depicted in the right side of Figure 3.

Finally, for the biomolecule in a salt solution the basic question is: “Is the saltattracted, or depleated from the vicinity of the biomolecule?” These situations areschematically drawn in Figure 2. For the quantification, the so called preferrentialbinding parameter, Γ, is introduced (Equation 1). This single number is verypowerful as it is directly related to the change of thermodynamic properties of abiomolecule in a given solution.

Γ =

⟨N vicinity

salt − N bulksalt

N bulkwater

N vicinitywater

⟩(1)

where the N localsalt , N bulk

salt are the number of salt molecules in the vicinity and farfrom the biomolecule, and the N local

water, N bulkwater represent the same for water. The

formula simply displays if the salt is more attracted (Γ > 0) or excluded (Γ < 0)from the vicinity of a biomolecule compared to its concentration in solution.

1.4 Results and discussion 8

Figure 2. Competition of water (blue spheres) and cosolvent (yellow ellipses)molecules for the vicinity of the protein (a large gray sphere). The preferentialbinding of cosolvent (excess of cosolvent, Γ > 0 – the left figure), and preferentialhydration (depletion of cosolvent, Γ < 0 – the right figure).

1.4 Results and discussion

This thesis consists of 13 articles in which we have investigated ion-specificeffects in solutions. We focus on interactions of ions with peptide and proteinsurfaces, nevertheless, the objects of our interest range from solutions of molec-ular salts (i.e., ammonium chloride, or guanidinium sulphate), salts acting onsmall organic fragments, or amino acids, to the effect on surfaces of peptides andproteins.

All these projects have shed more light on the following issues:

• Understanding of ion-specific effects, and consequent ordering in Hofmeisterseries [17,18] for cations, anions, and osmolytes.

• Interaction of ions with organic molecules and biomolecules.

• Rationalization of the reductionistic approach and understanding of its lim-itations.

To these points, my contribution, following the complexity of the system, canbe divided into three parts:

1. Interaction of ions with model systems. Examples are small molecules thatmimic the peptide bond or amino acid side chain.

2. Ion-amino acid interaction, with dominant, but not exclusive, focus on pos-itively charged amino acids.

3. Ion-peptide and ion-protein interaction. This part shows the diversity thatis a genuine property of complex systems.

It seems to be convenient to illustrate here on the molecule of N-methylacet-amide the concepts that were used in our work. Other systems are more difficultto introduce within the limited abstract format. The reader is referred to ourpublications for more details.

1.4 Results and discussion 9

Figure 3. Spatial distribution (left) of iodide (top, in violet) and chloride (bot-tom, in gold) anion and sodium cation (green) around N-methylacetamide com-plemented by partial contributions (of carbonyl (red), amide (blue), and methylgroups (black)) of the proximal distribution function (right).

1.4.1 Case study – Model of the peptide bond

In our case, the ultimate goal is to understand the interaction between ionsand proteins. The composition obviously varies, but the peptide bond is a uniquefeature that is always present. For that reason we investigated how the sodium/po-tassium halides solutions affect N-methylacetamide (NMA) – i.e., the proxy ofthe peptide bond. The simple and fairly rigid molecule is shown in the left part ofFigure 3. It is the peptide bond to some extent protected on both ends by methylgroups.

This model system is worth a deeper exploration mainly due to the recentresults of Bolen and Rose [19, 20]. It was shown that the peptide bond is thekey player for protein stabilization and denaturation. That is why the deeperknowledge about NMA properties in salt solutions is essential.

Owing to the fact that NMA contains a polar part (the peptide bond) and alsohydrophobic pieces (methyl groups) we conducted also polarizable simulations[15], which treat more accurately the weak, but important, interaction of anionswith hydrophobes.

Simulations were analyzed not only in terms of spatial, proximal and standardradial distribution functions, but also the distribution of residence times wasestimated together with mean times of interaction that were calculated based onthe first order kinetic assumptions.

The spatial distribution functions in Figure 3 clearly show that the polargroups are the most prominent sites for both cations (carbonyl oxygen) and an-ions (amide hydrogen). However, since the accessible volume is rather limited,the corresponding proximal distribution functions carry this preference only for

1.4 Results and discussion 10

cations (height of the peak is roughly 4 for sodium), while being around unity foranions. The second very important observation is the smeared, but in total strongand robust, preference of softer anions for the methyl groups (peaks ranging from∼1.5 for chloride to ∼3 for iodide). This fact leads to an exclusion of fluoride,neutral behaviour of chloride, and to attraction of bromide and iodide to NMA.

The results of other projects are briefly summarized in the next sections. Allthe projects are framed by the concept of ion-specific interactions.

1.4.2 Amino acid proxies

Starting with the simpler systems, we were able to interpret and proposea mechanism by which ions affect the peptide bond, finding the strong affinityof cations, but only modest of anions. This effect has direct implications forproteins [15].

To probe the Collins concept of matching water affinities [21] for ammoniumcation we employed the combined MC-MD approach [22] and compared with ex-perimental data available (through the activity coefficients of ammonium salts).We observed significantly stronger pairing of ammonium with small halides im-plying that the ammonium should be considered the small (so called hard) cation,in contrast to the concept of water affinities.

We touched the same topic using the neutron scattering experiments linkedwith MD [23] calculations, where we found that the ammonium and ammoniummoieties in lysine and zwitterionic glycine are not equally attractive for fluorideanion. On the contrary, iodide binds similarly in all cases.

1.4.3 Single amino acids and short oligopeptides

The positively charged amino acids (monomers) are perfect targets for halideanions. We characterized the organization of small (F−) and large (I−) anions inthe vicinity of positivey charged sidechains (arginine, lysine, histidine) [24]. Wefound not only that smaller anions bind in all cases stronger, but also that thebinding strength is sidechain-dependent (arginine histidine lysine).

The positively charged amino acids themselves already as dipeptides exhibit asurprising feature. Similarly to a pair of sodium cations one expect the repulsionof two sidechains of the like charge. However, we revealed and quantitativelycharacterized the origin of like-charge attraction (parallel stacking) in case of twoarginines and two histidines [25–27]. This effect does not exist for two lysines.

One always asks for a more direct proof of any qualitative effect. To that point,motivated by mounting evidence of specific behaviour of guanidinium (cation-cation stacking, strong pairing with sulphate, etc.), we succesfully combined theMD framework with measurements of electrophoretic mobilities of tetra-argineand tetra-lysine [28,29]. While MD provided the initial motivation and atomisticinsight, capillary electrophoresis provided hard experimental data, placing theproposed effects on solid ground.

In tandem with surface sensitive experimental techniques (second harmonicgeneration (SHG) spectroscopy) we explored the pH dependent surface activityof the β-amyloid 1-16 fragment, charge of which varies dramatically from +6 atpH=3, through +2 at pH=7 to -6 at pH=11. The results captured the dominant

1.5 Summary 11

role of total charge and sketched the surface behaviour of positively and negativelycharged groups [30].

1.4.4 Peptides and proteins

β-amyloid 1-16 fragment was a 16 amino acid long peptide. However ,it doesnot exhibit features of larger proteins, such as stable secondary structure ele-ments or three dimensional fold. The 20 amino acid long TrpCage minipeptidewas designed and synthetized in 2002 so as to mimic the large proteins [31].While other research groups concentrated on the thermodynamical description ofpeptide unfolding [32], we focused on the dynamics of peptide denaturation.

Two widely used denaturants (urea and guanidinium chloride) were investi-gated when acting on the TrpCage minipeptide [14]. Two different (denaturant-specific) pathways of unfolding were observed, however, with a similar result– i.e., the ensemble of denatured states. The denaturation processes were ex-perimentally characterized by three independent techniques (circular dichroism,differential scanning calorimetry and nuclear magnetic resonance).

The different denaturating effect of neighbouring cations in Hofmeister series[18] (tetrapropyl ammonium and guanidinium) was rationalized based on the pairinteractions. In this way we took into acount both the ion-specific interactions insalt solution and the ion-specific interaction with the protein surface [33].

The powerful denaturing action of guanidinium can be weakened by an ad-dition of sulphate, which is related to strong cation-anion pairing. In contrast,tetrapropyl ammonium is insensitive to anions, but being unable to create hydro-gen bonds it may be a denaturant for one and stabilizer for another protein. Thepredictions we made in 2009 came true in 2011, when they were experimentallyverified [34].

Even though Hofmeister measured the salting out effect of egg white protein[17], the Hofmeister series miraculously appear across fields. Revealing of theeffect of salts on enzymatic activity is still far from being reached, nevertheless,we have tried to tackle this problem.

The Hofmeister series for cations was tested in two studies employing very rel-evant enzymes. In the first case, the enzymatic activity assay (within Michaelis-Menten kinetics) of HIV-1 protease was performed in sodium chloride and potas-sium chloride, observing the activity consistently higher by 20% in the lattercase. MD calculations found generally a twice higher affinity of sodium cationto the enzyme surface, and on top of that, particular spatially resolved maximaaround the active site [13].

The second study, which is still under progress, targets the catalytic activityof the LinB dehalogenase, in a large set of salt solutions at various concentrations.From the computational side, we aimed at alkali chloride salts where we found toa large extent direct Hofmeister ordering [18].

1.5 Summary

To summarize, in this thesis I demonstrated the wide range of applications forMD simulations for biologically relevant systems at various level of complexity.The all-atom description allowed to obtain information about ion-specific effects

1.5 Summary 12

in all studied contexts. Due to the fact that proteins are always exposed to solventsand salty solutions this work has potential applications to many different fields,such as biophysics, biochemistry, biology or biotechnology.

2 Autoreferat v ceskem jazyce 13

2 Autoreferat v ceskem jazyce

2.1 Uvod do komplexnıch systemu

Biomolekuly lze jen tezko nalezt mimo vodne prostredı a to jen malokdyneobsahuje zadne ionty. Bılkovina ve slane vode je prıkladem tzv. komplexnıhosystemu, ktery byva asi nejcastejsım predmetem vyzkumu ve vypocetnı komu-nite [1–4]. Cetnost a kvalita publikacı jde ruku v ruce s poznanım, ktere jevyse ukazaly byt vyznamne a ktere nikoliv. Stava se tak cım dal obtıznejsı prijıts prulomovym objevem, jenz je jednım z nezbytnych predpokladu pro prijetıdo vysoce impaktovanych casopisu v oboru roztoku jednoduchych solı a neelek-trolytu.

Presto se i v poslednı dobe muzeme setkat se zasadnımi poznatky i protak jednoduchy system, jakym je cista voda. Voda je tak stale vdecne temavyzyvajıcı k dalsımu badanı. Napr. Laage [5] se zabyval tım, jak pri rotacıchmolekul vody dochazı k vzniku a zaniku vodıkove vazby, Sedlmeier [6] srovnalvysledky pocıtacovych simulacı ciste vody s experimenty neutronoveho rozptylu,a konecne Dzubiella [7] uvedl novy model pro vypocet implicitnı solvatace.

Jakmile je sledovany system, at jiz se jedna o polymer, peptid nebo pouzehydrofobnı kouli, umısten do roztoku, moznosti novych podnetu ke studiu vyraznenarostou [2, 8–10]. V poslednı dobe vsak jiz nejde pouze o to, zda efekt existuje,ale zejmena o sılu tohoto efektu. Vetsinou je to prave detailnı rovnovaha mezisouperıcımi interakcemi, ktera je klıcova v bio-systemech (napr. elektrostatickainterakce versus hydratacnı energie).

Bılkoviny jsou velmi rozmanite molekuly a z toho duvodu je pro ne tezkenalezt obecne platna pravidla. Nicmene zakladnı kameny – dvacet aminokyselin,ktere jsou spojovany peptidovou vazbou – jsou vzdy prıtomne. V nejhrubsımpriblızenı pak muzeme veskery vyznam pripsat jejich funkcnım skupinam uve-denym na obrazku 4, protoze to je to jedine, cım se lisı. Vse ostatnı zanedbame.To vede ke studiu nabitych skupin (karboxylova skupina, amonny kation, imidazo-liovy kation, guanidny kation), polarnıch skupin (alkoholy, thioly, karboxamidy) atake peptidove vazby. Bohuzel nebylo zatım dostatecne prokazano, zda je takovypostup opodstatneny a spravny.

Obrazek 4. Funkcnı skupiny aminokyselin, kterym je prisuzovan zasadnı vlivna lokalnı interakci s ionty a solventy. Nabita karboxylova skupina aspartatu,glutamatu a C-konce (A), amonna skupina lysinu, resp. N-konce (B), guanidnaskupina z postrannıho retezce argininu (C), imidazoliova skupina histidinu (D),alkoholy a thioly (E), karboxamidova skupina asparaginu a glutaminu (E), apeptidova vazba (H).

2.2 Cıle prace 14

Zasadnı zvrat nastal v roce 2007, kdy Auton, Holthauzen a Bolen [11] kom-pletnım zpusobem zrevidovali experimentalnı a teoreticka data ze 70. let. Jejichprace vnesla zcela nove svetlo do popisu denaturace proteinu, cımz znovu ob-jevili a prokazali platnost tzv. transferoveho modelu [12], ktery nenı nic jinehonez proces rozdelenı proteinu na jednotlive casti.

2.2 Cıle prace

Bylo by velice uzitecne, kdyby bylo realne studovat systemy a jejich vzajemneinterakce v jejich prirozenem prostredı, ale dıky jejich slozitosti je to zatım mimonase moznosti a musıme se proto uchylit k aproximacım. Nejbeznejsı je tzv.metoda redukce, kdy se puvodnı system rozseka na kousky a ty se studujı oddelenev ramci nejlepsı (a nejslibnejsı) dostupne teorie.

Z tohoto pohledu se jako nejvyhodnejsı jevı zacıt simulacemi interakcı iontumezi sebou, dale iontu a malych organickych molekul, a pote iontu a aminokyselin,cımz se konecne dostaneme ke kyzenym proteinum. I presto, ze dulezite jsouzejmena prvnı dva kroky, je z praktickych duvodu potreba postupovat soucasnena ruznych urovnıch.

V nasem prıpade jsme navazali na praci Vrbka et al. [2] a zabyvali se vlivemchloridu sodneho a draselneho na HIV-1 proteazu. Z vypocetnıho hlediska vy-sledky potvrdily preferenci sodnych kationtu nad draselnymi na povrchu HIV-1proteazy [13]. Podstatne bylo, ze jsme se setkavali s dılcımi jevy, ktere je trebapochopit, abychom mohli plne porozumet problematice proteinu v cele jejı sıri.

Projekty zkoumajıcı proteiny byly dlouhodobeho charakteru, nebot pred nasopakovane kladly nove prekazky a vyzvy (at fundamentalnı ci technicke) [13,14].Tım vsak s sebou prinasely mensı projekty, ktere byly zajımave per-se.

2.3 Vypocetnı metody

2.3.1 Simulacnı protokol

Klasicka molekulova dynamika se ukazala jako idealnı volba pro studiumnasich systemu. Casto bylo podstatne pouze zıskat sadu struktur, avsak v ne-kterych prıpadech byl dulezity casovy vyvoj [14, 15]. K tom dochazelo tehdy,kdyz byla studovana prımo dynamika daneho deje.

Na zacatku kazdeho projektu stojı prıprava systemu. Pokusım se nastınitdulezite kroky. Nejprve se molekuly vody a soli nahodne smısı v pomeru, kteryodpovıda cılove koncentraci. Nasledne provedeme kratkou minimalizaci energiesystemu, jez nas zbavı nezadoucıch artefaktu (napr. nahodneho prekryvu dvouatomu apod.). Prakticky vzdy pouzıvame v simulaci periodicke okrajove podmın-ky, tj. rozmnozıme nas system ve vsech rozmerech, cımz dostaneme nekonecnysystem. Jde o dulezity a bezne pouzıvany trik, jak ze systemu o cca 1000 ato-mech udelat obrovsky system, kde jiz beze zbytku platı zakony statistiky. Nazaver system zahrejeme a stlacıme, aby mel pozadovanou hustotu a teplotu.

Pokud chceme studovat chovanı molekuly vystavene ucinkum slaneho roz-toku, vlozıme ji do pripraveneho systemu a nechame ho vyvıjet v case, cımzprozkoumame zejmena relevantnı (ale do jiste mıry i jine malo pravdepodobne)

2.4 Vysledky a diskuse 15

stavy systemu. Ty vytvorı statisticky soubor, ktery muzeme nasledne analyzovat,naprıklad metodami popsanymi v nasledujıcı kapitole.

Je treba jeste podotknout, ze casovy krok pouzıvany v simulacıch je velmikratky – typicky 1 fs – a veskery pohyb je rızen Newtonovymi rovnicemi. Jed-notlive castice na sebe pusobı tzv. silovym polem, coz je predem stanoveny efek-tivnı potencial (sada interakcnıch parametru).

2.3.2 Druhy analyzy

Pro rutinnı analyzy jsme pouzıvali volne dostupny modul PTRAJ [16]. Du-lezitym prıkladem je tzv. radialnı distribucnı funkce (RDF), ktera vyjadrujepravdepodobnost nalezenı dvou objektu (naprıklad kationtu a aniontu) v danevzdalenosti. Jejı zasadnı vyznam pramenı ze skutecnosti, ze se jedna o ustrednıvelicinu ve statisticke termodynamice, prımo souvisejıcı s termodynamikou sys-temu. Abychom porozumeli iontove specifickym interakcım, potrebujeme mıt kdispozici typy analyzy, ktere jsou dostatecne popisne a zaroven i kvantitativnı.Tyto pokrocilejsı, mene obvykle metody byly implementovany v programovacımjazyku Python. Dve z nich, mapa pravdepodobnosti a tzv. proximalnı distribucnıfunkce, jsou popsany nıze.

• Mapa pravdepodobnosti poskytuje prostorove rozlozenı jednoho typu castickolem druheho umısteneho ve stredu. Pro nesfericke molekuly tak nesevyrazne vıce informace nez klasicky pouzıvana RDF. Prıklad jejıho pouzitıje v leve casti obrazku 6.

• Proximalnı distribucnı funkce je vyznamove podobna RDF, ale na rozdıl odnı bere do uvahy i tvar molekuly (proximalnı, tj. nejblıze umısteny), kolemktere je distribucnı funkce pocıtana. Prıklad jejıho pouzitı je znazornen vprave casti obrazku 6.

Ustrednı otazkou, kterou si klademe pri studiu biomolekul v roztocıch je, zdaje v jejı blızkosti slozenı roztoku odlisne cisteho roztoku. Na obrazku 5 jsou proilustraci zobrazeny tyto dve krajnı moznosti. Pro kvantifikaci tohoto efektu jezaveden tzv. preferencnı vazebny parametr Γ (rovnice 2),

Γ =

⟨N vicinity

salt − N bulksalt

N bulkwater

N vicinitywater

⟩(2)

kde N localsall , N bulk

salt vyjadruje pocet molekul soli (kosolventu) v blızkosti a dalekood biomolekuly, a N local

water, N bulkwater ma stejny vyznam, avsak pro vodu (obecneji

solvent). Hodnota parametru ma prımocary vyznam; zda se sul vyskytuje vıce(Γ > 0), nebo mene (Γ < 0) v blızkosti biomolekuly, nez odpovıda situacidaleko v roztoku.

2.4 Vysledky a diskuse

Tato doktorska prace sestava z 13 clanku v impaktovanych casopisech, v nichzjsme studovali iontove specificke efekty v nejruznejsıch prostredıch. Zacali jsme

2.4 Vysledky a diskuse 16

Obrazek 5. Kompetice molekul solventu (napr. vody; cervene krouzky) a ko-solventu (napr. soli; zlute elipsy) o okolı proteinu (velky sedy kruh). Nadbytekkosolventu (Γ > 0 – vlevo) a nadbytek molekul solventu (vyloucenı kosolventu,Γ < 0 – vpravo).

roztoky molekularnıch solı (jako naprıklad chloridem amonnym nebo guanidnym),pokracovali simulacemi aminokyselin vystavenych slanym roztokum, abychom senakonec zamerili na povrchy peptidu a proteinu.

Vsechny projekty mely prispet k hlubsımu porozumenı nasledujıcıch bodu:

• Iontove specificke efekty a jejich projev v Hofmeisterovych radach [17, 18]kationtu, aniontu a osmolytu.

• Interakce iontu a organickych molekul a biomolekul.

• Racionalizace metody redukce a stanovenı hranic jejı platnosti.

Muj prıspevek lze rozdelit do nasledujıcıch trı kapitol podle jejich rostoucıslozitosti:

1. Interakce iontu v modelovych systemech. Prıkladem budiz molekula napo-dobujıcı peptidovou vazbu nebo postrannı retezec aminokyseliny.

2. Interakce iontu s aminokyselinami, zejmena pak kladne nabitymi.

3. Interakce iontu a bılkovin. Tato cast ukazuje velkou rozmanitost, ktera jevlastnı prave komplexnım systemum.

Na molekule N-metylacetamidu je vhodne ukazat, jake postupy jsme obvyklepouzıvali pri nası praci. Ostatnı projekty jsou svou povahou komplikovanejsı nastrucne shrnutı, proto odkazuji laskaveho ctenare na nase publikace, kde muzenalezt vsechny detaily.

2.4.1 Prıklad – studie modelu peptidove vazby

Nasım cılem je porozumet interakcı mezi ionty a proteiny. Zamyslıme-li se nadtım, co je to protein, zjistıme, ze zatımco slozenım (tj. pocty ruznych aminokyselinv sekvenci) se jednotlive proteiny mezi sebou lisı, peptidova vazba je unikatnıprvek, ktery je vzdy prıtomny (prave jednou za kazdou aminokyselinu). Protojsme studovali, jak sodne a draselne soli halogenidu budou ovlivnovat N-metyl-acetamid (NMA), molekulu majıcı charakter peptidove vazby. Tato pomerne

2.4 Vysledky a diskuse 17

Obrazek 6. Mapa pravdepodobnosti (vlevo) jodidovych (nahore, ve fialovebarve) a chloridovych aniontu (dole, zlatou barvou) a sodnych kationtu (zele-nou barvou) kolem molekuly N-metylacetamidu vyjadrena komplementarne tremiprıspevky proximalnı distribucnı funkce (vpravo; karbonylove skupiny (v cervenebarve), amidove skupiny (v modre barve) a metylove skupiny (v cerne barve)).

rigidnı molekula s jednoduchou strukturou, zobrazena v leve casti obrazku 6,je v podstate peptidovou vazbou metylovanou na obou stranach.

System si zaslouzı blizsı pozornost take proto, ze nedavne vysledky Bolenaa Rose [19, 20] silne naznacujı, ze peptidova vazba je klıcovym elementem prostabilitu a denaturaci. I proto je poznanı vlastnostı NMA v roztocıch solı takdulezita.

Jelikoz NMA nese krome polarnı peptidove vazby i hydrofobnı metylove sku-piny, bylo treba do nasich vypoctu zahrnout polarizovatelnost vody a aniontu.Jenom tak bylo mozne spravne vyhodnotit sılu interakce aniontu s hydrofobnımiskupinami.

Data zıskana ze simulacı byla zpracovana pomocı map pravdepodobnosti,proximalnıch distribucnıch funkcı a klasickych RDF (tj. analyza, kde cas nenıdulezity). Dale jsme charakterizovali delku techto kontaktu za predpokladu, zeionty se k peptidove vazbe vazou kinetikou prvnıho radu (tj. explicitnı cas je zdedulezity).

Mapy pravdepodobnosti na obrazku 6 zcela jasne ukazujı, ze nejpravdepodob-nejsı pozice v okolı molekuly NMA jsou pro kationty v blızkosti karbonylovehokyslıku (ktery nese parcialnı zaporny naboj) a pro anionty u vodıku (parcialnıkladny naboj) amidove skupiny. Druhou nemene dulezitou skutecnostı je, ze pros-tor, ktery prıslusı temto energeticky vyhodnym lokacım, je prostorove vymezeny,zejmena pak pro anionty. Proto pri prechodu k proximalnı distribucnı funkcividıme (vpravo), ze relativne vysoky pık ma pouze sodny kation (cca o vysce 4),nikoliv tak anionty (kolem 1). Tretım faktem je, ze anionty (na rozdıl od kationtu)

2.4 Vysledky a diskuse 18

vykazujı afinitu k metylovym skupinam, kde krivka dosahuje hodnoty cca 1.5 prochloridovy a vıce nez 3 pro jodidovy aniont.

Vysledky ostatnıch projektu jsou shrnuty v dalsı kapitole. Muzeme v nichnajıt jednoho spolecneho jmenovatele – iontove specificke interakce.

2.4.2 Modely funkcnıch skupin

V predchozı kapitole jsme zacali jednoduchou molekulou, na nız jsme dokazalipredpovedet a interpretovat sılu a mechanismus interakce, kterym budou iontypusobit na peptidovou vazbu. Tato pozorovanı majı prıme dusledky pro vlastnostibılkovin ve slanych roztocıch [15].

Abychom overili koncept ’matching water affinities’ predpovezeny Collinsem[21], detailne jsme kombinacı molekulove dynamiky a metody Monte Carlo pro-zkoumali vazebne preference amonneho kationtu, kdyz se nachazı v roztoku shalogenidovymi anionty [22]. Vysledky jasne ukazaly, ze amonny kation preferujemale anionty pred vetsımi, a je tudız tzv. malym (nebo tez tvrdym) kationtem.Takovy vysledek je v rozporu s konceptem ’matching water affinities,’ ale je plnev souladu s namerenymi aktivitnımi koeficienty.

Toto tema jsme studovali take v kombinaci neutronoveho rozptylu a MD si-mulacı [23]. Vysledky pregnantne ukazujı, ze amonny kation a amonne skupinylysinu a glycinu nejsou k fluoridovemu aniontu pritahovany se stejnou intenzitou.Naopak jodid se vaze podobne ve vsech prıpadech.

2.4.3 Jednotlive aminokyseliny a kratke oligopeptidy

Kladne nabite aminokyseliny (v podobe monomeru) predstavujı pro halo-genidove anionty idealnı terc [24]. Charakterizovali jsme usporadanı malych flu-oridovych a velkych jodidovych aniontu v okolı kladne nabitych bocnıch retezcu(argininu, lysinu, histidinu). Mensı anionty se vazou silneji ve vsech prıpadech anavıc se ukazalo, ze vazebna sıla je zavisla na typu bocnıho retezce.

Kladne nabite aminokyseliny samy o sobe vykazujı zajımavou vlastnost. Stejnejako je samozrejme, ze se odpuzujı dva sodne kationty, je stejne tak prekvapive, zedva shodne nabite postrannı retezce argininu, nebo histidinu se pritahujı a tvorıparalelnı ’stack’. [25–27] Naproti tomu postrannı retezce lysinu se chovajı ’radne’a odpuzujı se.

Pro kazdy kvalitativnı jev, ktery je svym zpusobem neobvykly, je vhodnenajıt experiment, jenz jej dokaze i kvantifikovat. V nasem prıpade se ukazaloefektivnı pouzitı merenı elektroforeticke mobility pro peptidy tetraarginin a tetra-lysin [28, 29] v prıtomnosti solı obsahujıcı jedno- a dvoumocne anionty. ZatımcoMD prispela k motivaci vyzkumu a poskytla vhled na atomarnı urovni, kapilarnıelektroforeza namerila potrebna experimentalnı data a premenila efekty z hy-potetickych na realne.

Spolu s povrchove citlivou spektroskopickou metodou (generovanı druhe har-monicke frekvence (SHG)) jsme prozkoumali zavislost povrchove aktivity β-amyl-oid 1-16 fragmentu na pH. β-amyloid 1-16 fragment ma mnoho titrovatelnychskupin, a proto se jeho naboj v kyselem (+6), bazickem (-6) a neutralnım (+2)pH velmi lisı. Molekulova dynamika spravne zachytila dominantnı roli celkoveho

2.5 Zaver 19

naboje a nastınila mozne duvody pro pozorovane rozdıly v kyselem a bazickempH [30].

2.4.4 Peptidy a proteiny

β-amyloid 1-16 fragment ma 16 aminokyselin, presto nevykazuje chovanı vet-sıch proteinu, napr. nenajdeme u nej stabilnı sekundarnı strukturnı prvky nebobalenı do dominantnı tercialnı struktury. Oproti tomu v roce 2002 umele navrzeny20 aminokyselinovy TrpCage minipetid vsechny tyto rysy nese [31]. Zatımcoostatnı vedecke tymy se zamerily na popis termodynamickych vlastnostı jehosbalenı a rozbalenı [32], my jsme si dali za cıl charakterizovat proces denaturace.

Ucinek dvou nejbeznejsıch denaturantu (mocoviny a chloridu guanidneho) bylzkouman na minipeptidu TrpCage [14]. Prekvapive jsme zjistili, ze prubeh dena-turace je zavisly na denaturantu, nikoliv vsak vysledny efekt, ktery je stejny –TrpCage minipeptid v rozbalenem, denaturovanem stavu. Denaturace minipep-tidu byla nezavisle promerena tremi separatnımi technikami (cirkularnım dichro-ismem, kalorimetricky a pomocı nuklearnı magneticke rezonance).

V nasledujıcı studii jsme se zamerili na sousedy v Hofmeisterove rade ka-tiontu [18] – tetrapropylamonium a guanidinium. K vysvetlenı efektu jsme pouzilivsechny dosud zname efektivnı parove interakce, ktere vykazujı specificitu jakmezi samotnymi ionty v roztoku, tak i mezi ionty a povrchem proteinu [33].

Jedine v dusledku silneho kation-aniontoveho parovanı muze byt vysvetleno,proc se z velmi silneho denaturantu, kterym je chlorid guanidny, stane prumernydenaturant sıranu guanidneho. Naopak tetrapropyl amonium nenı vubec citlivena druh aniontu, s nımz se nachazı v roztoku, zaroven ale vzhledem k absencimoznosti tvorby vodıkovych vazeb muze byt pro jeden typ proteinu velmi silnymdenaturantem a pro druhe slabe stabilizujıcım osmolytem. Konecne nase predpo-vedi z roku 2009 se vyplnily po provedenı experimentu v roce 2011 [34].

Prestoze Hofmeister v roce 1888 seradil soli podle jejich schopnosti vysolovatvajecny bılek [17] z roztoku, objevujı se Hofmeisterovy rady jako zazrakem naprıcruznymi obory. Porozumet tomu, proc nektere soli zvysujı enzymatickou aktivitu,je beh na dlouhou trat, ale i tak se musı podstoupit.

Hofmeisterova rada pro kationty byla testovana ve dvou studiıch zabyvajıcıchse vybranymi enzymy. V prvnı z nich byl proveden rozbor enzymaticke aktivity(s pouzitım kinetiky Michaelis-Mentenove) HIV-1 proteazy v chloridu sodnem adraselnem, ktery v prıpade KCl roztoku vedl k objevu o 20% vyssı aktivity nez uNaCl. MD simulace poskytovaly obecne dvojnasobnou afinitu sodneho kationtuk povrchu enzymu a nadto ukazovaly na prostorove rozlisena maxima v okolıaktivnıho mısta.

Druha studie, jez je zatım ve fazi prıprav, ma za cıl poznanı charakteru kataly-ticke aktivity LinB dehalogenazy pro sadu roztoku solı o ruznych koncentracıch.Z vypocetnıho hlediska jsme se zamerili na chloridy alkalickych kovu, u kterychjsme do znacne mıry pozorovali Hofmeisterovo usporadanı [18].

2.5 Zaver

Zaverem bych uvedl, ze v predkladane dizertacnı praci bylo prezentovanosiroke spektrum aplikacı molekulove dynamickych simulacı, a to zejmena pro

2.5 Zaver 20

biologicky relevantnı systemy s ruznym stupnem komplexity. Popis na urovnijednotlivych atomu a molekul nam umoznil zıskat poznatky o iontove specifickychefektech ve vsech studovanych kontextech. Dıky skutecnosti, ze proteiny jsou vesvem prirozenem prostredı vzdy vystaveny pusobenı solventu a roztoku solı, matato prace potencialnı vyuzitı v mnoha vednıch oborech, jako napr. v biofyzice,biochemii, biologii nebo biotechnologii.

REFERENCES 21

References

[1] G. Hummer, S. Garde, A. E. Garcia, A. Pohorille, and L. R. Pratt, “An information theorymodel of hydrophobic interactions”, Proceedings of the National Academy of Sciences ofthe United States of America 93(17), pp. 8951–8955 (1996).

[2] L. Vrbka, J. Vondrasek, B. Jagoda-Cwiklik, R. Vacha, and P. Jungwirth, “Quantificationand rationalization of the higher affinity of sodium over potassium to protein surfaces”,Proceedings of the National Academy of Sciences of the United States of America 103(42),pp. 15440–15444 (2006).

[3] G. D. Rose, P. J. Fleming, J. R. Banavar, and A. Maritan, “A backbone-based theory ofprotein folding”, Proceedings of the National Academy of Sciences of the United States ofAmerica 103(45), pp. 16623–16633 (2006).

[4] D. Horinek, A. Serr, M. Geisler, T. Pirzer, U. Slotta, S. Q. Lud, J. A. Garrido, T. Scheibel,T. Hugel, and R. R. Netz, “Peptide adsorption on a hydrophobic surface results froman interplay of solvation, surface, and intrapeptide forces”, Proceedings of the NationalAcademy of Sciences of the United States of America 105(8), pp. 2842–2847 (2008).

[5] D. Laage and J. T. Hynes, “A molecular jump mechanism of water reorientation”, Science311(5762), pp. 832–835 (2006).

[6] F. Sedlmeier, D. Horinek, and R. R. Netz, “Spatial correlations of density and structuralfluctuations in liquid water: A comparative simulation study”, Journal of the AmericanChemical Society 133(5), pp. 1391–1398 (2011).

[7] J. Dzubiella, J. M. J. Swanson, and J. A. McCammon, “Coupling hydrophobicity, dis-persion, and electrostatics in continuum solvent models”, Physical Review Letters 96(8)(2006).

[8] G. Hummer, S. Garde, A. E. Garcia, M. E. Paulaitis, and L. R. Pratt, “The pressuredependence of hydrophobic interactions is consistent with the observed pressure denatu-ration of proteins”, Proceedings of the National Academy of Sciences of the United Statesof America 95(4), pp. 1552–1555 (1998).

[9] D. Chandler, “Interfaces and the driving force of hydrophobic assembly”, Nature 437, pp.640–647 (2005).

[10] D. Horinek, A. Herz, L. Vrbka, F. Sedlmeier, S. I. Mamatkulov, and R. R. Netz, “Specificion adsorption at the air/water interface: The role of hydrophobic solvation”, ChemicalPhysics Letters 479(4-6), pp. 173–183 (2009).

[11] M. Auton, L. M. F. Holthauzen, and D. W. Bolen, “Anatomy of energetic changes ac-companying urea-induced protein denaturation”, Proceedings of the National Academy ofSciences of the United States of America 104, pp. 15317–15322 (2007).

[12] Y. Nozaki and C. Tanford, “Solubility of amino acids and related compounds in aqueousurea solutions”, Journal of Biological Chemistry 238(12), pp. 4074– (1963).

[13] J. Heyda, J. Pokorna, L. Vrbka, R. Vacha, B. Jagoda-Cwiklik, J. Konvalinka, P. Jungwirth,and J. Vondrasek, “Ion specific effects of sodium and potassium on the catalytic activityof HIV-1 protease”, Physical Chemistry Chemical Physics 11(35), pp. 7599–7604 (2009).

[14] J. Heyda, M. Kozısek, L. Bednarova, G. Thompson, J. Konvalinka, J. Vondrasek, andP. Jungwirth, “Urea and guanidinium induced denaturation of a trp-cage miniprotein”,The Journal of Physical Chemistry B (2011), doi: 10.1021/jp200790h.

[15] J. Heyda, J. C. Vincent, D. J. Tobias, J. Dzubiella, and P. Jungwirth, “Ion specificity atthe peptide bond: Molecular dynamics simulations of n-methylacetamide in aqueous saltsolutions”, Journal of Physical Chemistry B 114(2), pp. 1213–1220 (2010).

REFERENCES 22

[16] T.E. Cheatham III C.L. Simmerling J. Wang R.E. Duke R. Luo R.C. Walker W. ZhangK.M. Merz B. Roberts B. Wang S. Hayik A. Roitberg G. Seabra I. Kolossvai K.F. WongF. Paesani J. Vanıcek J. Liu X. Wu S.R. Brozell T. Steinbrecher H. Gohlke Q. Cai X. YeJ. Wang M.-J. Hsieh G. Cui D.R. Roe D.H. Mathews M.G. Seetin C. Sagui V. Babin T.Luchko S. Gusarov A. Kovalenko D.A. Case, T.A. Darden and P.A. Kollman, “Amber 11”(2010).

[17] F. Hofmeister, “Zur lehre von der wirkung der salze - zweite mittheilung”, Archiv furExperimentelle Pathologie und Pharmakologie 24(4-5), pp. 247–260 (1888).

[18] W. Kunz, J. Henle, and B. W. Ninham, “’zur lehre von der wirkung der salze’ (about thescience of the effect of salts): Franz hofmeister’s historical papers”, Current Opinion inColloid & Interface Science 9(1-2), pp. 19–37 (2004).

[19] Y. F. Liu and D. W. Bolen, “The peptide backbone plays a dominant role in proteinstabilization by naturally-occurring osmolytes”, Biochemistry 34(39), pp. 12884–12891(1995).

[20] T. O. Street, D. W. Bolen, and G. D. Rose, “A molecular mechanism for osmolyte-inducedprotein stability”, Proceedings of the National Academy of Sciences of the United Statesof America 103(38), pp. 13997–14002 (2006).

[21] Kim D. Collins, “Ions from the hofmeister series and osmolytes: effects on proteins insolution and in the crystallization process”, Methods 34(3), pp. 300–311 (2004).

[22] J. Heyda, M. Lund, M. Oncak, P. Slavıcek, and P. Jungwirth, “Reversal of hofmeisterordering for pairing of NH+

4 vs alkylated ammonium cations with halide anions in water”,Journal of Physical Chemistry B 114(33), pp. 10843–10852 (2010).

[23] P. E. Mason, J. Heyda, H. E. Fischer, and P. Jungwirth, “Specific interactions of ammo-nium functionalities in amino acids with aqueous fluoride and iodide”, Journal of PhysicalChemistry B 114(43), pp. 13853–13860 (2010).

[24] J. Heyda, T. Hrobarik, and P. Jungwirth, “Ion-specific interactions between halides andbasic amino acids in water”, Journal of Physical Chemistry A 113(10), pp. 1969–1975(2009).

[25] J. Vondrasek, P. E. Mason, J. Heyda, K. D. Collins, and P. Jungwirth, “The molecularorigin of like-charge arginine-arginine pairing in water”, Journal of Physical Chemistry B113(27), pp. 9041–9045 (2009).

[26] J. Heyda, P. E. Mason, and P. Jungwirth, “Attractive interactions between side chains ofhistidine-histidine and histidine-arginine-based cationic dipeptides in water”, Journal ofPhysical Chemistry B 114(26), pp. 8744–8749 (2010).

[27] Mario Vazdar, Jirı Vymetal, Jan Heyda, Jirı Vondrasek, and Pavel Jungwirth, “Like-chargeguanidinium pairing from molecular dynamics and ab initio calculations”, The Journal ofPhysical Chemistry A (2011), doi: 10.1021/jp203519p.

[28] E. Wernersson, J. Heyda, A. Kubıckova, T. Krızek, P. Coufal, and P. Jungwirth, “Effectof association with sulfate on the electrophoretic mobility of polyarginine and polylysine”,Journal of Physical Chemistry B 114(36), pp. 11934–11941 (2010).

[29] Anna Kubıckova, Tomas Krızek, Pavel Coufal, Erik Wernersson, Jan Heyda, and PavelJungwirth, “Guanidinium cations pair with positively charged arginine side chains inwater”, The Journal of Physical Chemistry Letters 2(12), pp. 1387–1389 (2011).

[30] A. E. Miller, P. B. Petersen, C. W. Hollars, R. J. Saykally, J. Heyda, and P. Jungwirth,“Behavior of beta-amyloid 1-16 at the air-water interface at varying ph by nonlinear spec-troscopy and molecular dynamics simulations”, Journal of Physical Chemistry A 115(23),pp. 5873–5880 (2011).

[31] J. W. Neidigh, R. M. Fesinmeyer, and N. H. Andersen, “Designing a 20-residue protein”,Nature Structural Biology 9(6), pp. 425–430 (2002).

REFERENCES 23

[32] D. R. Canchi and A. E. Garcia, “Backbone and side-chain contributions in protein denat-uration by urea”, Biophysical Journal 100(6), pp. 1526–1533 (2011).

[33] P. E. Mason, C. E. Dempsey, L. Vrbka, J. Heyda, J. W. Brady, and P. Jungwirth, “Speci-ficity of ion-protein interactions: Complementary and competitive effects of tetrapropy-lammonium, guanidinium, sulfate, and chloride ions”, Journal of Physical Chemistry B113(10), pp. 3227–3234 (2009).

[34] Christopher E. Dempsey, Philip E. Mason, and Pavel Jungwirth, “Complex ion effectson polypeptide conformational stability: Chloride and sulfate salts of guanidinium andtetrapropylammonium”, Journal of the American Chemical Society 133(19), pp. 7300–7303 (2011).

Curriculum vitae

Name: Jan HeydaBorn: 18th June 1983 in Cesky KrumlovEmail: jan.heyda@uochb.cas.czAddress: Lipovska 436, Praha 5 – Zlicın, 155 21

Education:

• 2007-Faculty of Science, Charles University in Prague, study program: PhysicalChemistry; Modelling of Chemical Properties of Nano- and BiostructuresPhD project: Ion-protein interactionsSupervisor: Prof. Mgr. Pavel Jungwirth, DSc.

• 2007-Member of the International Max Planck Research School, Dresden“Dynamical Processes in Atoms, Molecules and Solids”

• 2002-2008Faculty of Mathematics and Physics, Charles University in Prague, studyprogram: Mathematics; Mathematical Modeling in Physics and EngineeringDiploma Thesis: Distribution of ions at surfaces of hydrated proteinsSupervisor: Doc. Mgr. Pavel Jungwirth, CSc.

• 2002-2007Faculty of Science, Charles University in Prague, study program: PhysicalChemistry; group: Electromigration and Separation MethodsDiploma Thesis: Multidimensional Simulation of ElectromigrationSupervisor: Prof. RNDr. Bohuslav Gas, CSc.

Work Experience:

• Since October 2006 -Academy of Sciences of the Czech Republic, Institute of Organic Chemistryand Biochemistry

• Since January 2006 – June 2008Work in program COMSOL Multiphysics with respect to numerical solutionof partial-differential equations

• March 2007 – June 2008Basic Research Project with Agilent Technologies

Selected Publications/Seznam publikacı

1. J. Heyda, T. Hrobarik, and P. Jungwirth, “Ion-specific interactions between halides andbasic amino acids in water”, Journal of Physical Chemistry A 113(10), pp. 1969–1975(2009).

2. P. E. Mason, C. E. Dempsey, L. Vrbka, J. Heyda, J. W. Brady, and P. Jungwirth, “Speci-ficity of ion-protein interactions: Complementary and competitive effects of tetrapropy-lammonium, guanidinium, sulfate, and chloride ions”, Journal of Physical Chemistry B113(10), pp. 3227–3234 (2009).

3. J. Vondrasek, P. E. Mason, J. Heyda, K. D. Collins, and P. Jungwirth, “The molecularorigin of like-charge arginine-arginine pairing in water”, Journal of Physical ChemistryB 113(27), pp. 9041–9045 (2009).

4. J. Heyda, J. Pokorna, L. Vrbka, R. Vacha, B. Jagoda-Cwiklik, J. Konvalinka, P. Jung-wirth, and J. Vondrasek, “Ion specific effects of sodium and potassium on the catalyticactivity of HIV-1 protease”, Physical Chemistry Chemical Physics 11(35), pp. 7599–7604(2009).

5. J. Heyda, J. C. Vincent, D. J. Tobias, J. Dzubiella, and P. Jungwirth, “Ion specificityat the peptide bond: Molecular dynamics simulations of N-methylacetamide in aqueoussalt solutions”, Journal of Physical Chemistry B 114(2), pp. 1213–1220 (2010).

6. J. Heyda, P. E. Mason, and P. Jungwirth, “Attractive interactions between side chainsof histidine-histidine and histidine-arginine-based cationic dipeptides in water”, Journalof Physical Chemistry B 114(26), pp. 8744–8749 (2010).

7. J. Heyda, M. Lund, M. Oncak, P. Slavıcek, and P. Jungwirth, “Reversal of hofmeisterordering for pairing of NH+

4 vs alkylated ammonium cations with halide anions in water”,Journal of Physical Chemistry B 114(33), pp. 10843–10852 (2010).

8. E. Wernersson, J. Heyda, A. Kubıckova, T. Krızek, P. Coufal, and P. Jungwirth, “Effectof association with sulfate on the electrophoretic mobility of polyarginine and polylysine”,Journal of Physical Chemistry B 114(36), pp. 11934–11941 (2010).

9. P. E. Mason, J. Heyda, H. E. Fischer, and P. Jungwirth, “Specific interactions of am-monium functionalities in amino acids with aqueous fluoride and iodide”, Journal ofPhysical Chemistry B 114(43), pp. 13853–13860 (2010).

10. Anna Kubıckova, Tomas Krızek, Pavel Coufal, Erik Wernersson, Jan Heyda, and PavelJungwirth, “Guanidinium cations pair with positively charged arginine side chains inwater”, The Journal of Physical Chemistry Letters 2(12), pp. 1387–1389 (2011).

11. A. E. Miller, P. B. Petersen, C. W. Hollars, R. J. Saykally, J. Heyda, and P. Jungwirth,“Behavior of beta-amyloid 1-16 at the air-water interface at varying pH by nonlinearspectroscopy and molecular dynamics simulations”, Journal of Physical Chemistry A115(23), pp. 5873–5880 (2011).

12. J. Heyda, M. Kozısek, L. Bednarova, G. Thompson, J. Konvalinka, J. Vondrasek, andP. Jungwirth, “Urea and guanidinium induced denaturation of a trp-cage miniprotein”,The Journal of Physical Chemistry B (2011), doi: 10.1021/jp200790h.

13. Mario Vazdar, Jirı Vymetal, Jan Heyda, Jirı Vondrasek, and Pavel Jungwirth, “Like-charge guanidinium pairing from molecular dynamics and ab initio calculations”, TheJournal of Physical Chemistry A (2011), doi: 10.1021/jp203519p.