Thorn Leeson, Daniël

Wiersma, D. A.

English

Dissertations of the University of Groningen

   University of GroningenExploring protein energy landscapesThorn Leeson, Daniël; Wiersma, D. A.IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1997Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Thorn Leeson, D., & Wiersma, D. A. (1997). Exploring protein energy landscapes. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022GENERAL INTRODUCTION11GENERAL INTRODUCTION1.1 Protein structure and dynamicsProteins are indispensable elements in the machinery of life. [Str88] They contribute tothe functioning of living systems in a multitude of ways. For instance, proteins supply thestructural basis to fibrous tissue and muscle, are involved in the transport of small molecules,electrons, and energy, and catalyze chemical reactions. The human body alone, contains anestimate of 100,000 different proteins, each of which has a unique function. A particularprotein is characterized by the sequence of the amino acids in the polypeptide chain, whichis referred to as its primary structure. It is this sequence that determines the tertiary structureof a protein, which is the intricate three-dimensional arrangement of its atoms in the nativestate. Although each protein has a unique, well-defined tertiary structure, often regularlyrepeating conformations such as "-helices, $-sheets, and $-turns are observed. Thesestructural elements comprise the so-called secondary structure of a protein.The first protein to have its three-dimensional structure revealed was myoglobin, a smallglobular protein which has the function of transporting dioxygen in muscle tissue. In 1960,Kendrew et al. [Ken60] presented a structural model of myoglobin with a resolution of 2 Å,based on x-ray diffraction patterns. The three-dimensional structure of my globin isdisplayed in Fig. 1.1. Myoglobin is a very compact protein, consisting mainly of "-helix. Itsdimensions are roughly 45 × 35 × 25 Å, and it has a molecular weight of approximately 17kD. Myoglobin contains 153 amino acids. Furthermore, it contains a heme group, which isthe binding site for oxygen. The exploratory work of Kendrew et al. marked a milestone inthe field of structural biology, and since then the structures of an ever growing number ofproteins have been resolved. The widespread interest in protein structure is based on the common view that structureand biochemical function are intimately related and many examples are available that supportthe general existence of such a relationship. When the first tertiary structures were beingEXPLORING PROTEIN ENERGY LANDSCAPES2Figure 1.1  Structural model of myoglobin. The central heme is recognizable by its lightshading.resolved, proteins were generally conceived of as rigid bodies. Earlier on, Schrödinger[Sch44] referred to them as aperiodic crystals, aperiodic in the sense that they do not exhibita regularly repeating structural pattern like ordinary crystals, but crystalline in the sense thatthe position of each atom is fixed at a well defined point in space. However, experimentaland computational evidence is rapidly accumulating that shows that this view needs to beadjusted. Proteins exhibit a multitude of motions ranging from local atomic fluctuations tocomplex global rearrangements. [Bro88] As a consequence, proteins exhibit structuralheterogeneity. Therefore, the atomic coordinates obtained from x-ray diffraction and nuclearmagnetic resonance studies should be considered as averages, either ensemble averages, ortime averages of single proteins. This combination of crystalline properties and propertiesthat are associated with disordered condensed phases, such as glasses and liquids, qualifiesproteins as a unique state of matter.A protein consists of both rigid and flexible groups of atoms. A substantial contributionto the conformational flexibility of a protein arises from fluctuations of the polypeptideGENERAL INTRODUCTION3backbone. The polypeptide chain is made up of fairly rigid groups, the CONH units, linkedby carbon-carbon single bonds that allow rotations. Substantial displacements arise fromtorsional motions around these single bonds. Although low amplitude fluctuations may occurindependently on an atomic level, larger scale rearrangements are expected to occur in aconcerted way. This is because proteins are densely packed, which implies spatial restrictionsin the sense that for one atom or groups of atoms to move, neighboring groups have to giveway. Since the dense packing is stabilized by energetically favorable interactions, such ashydrogen bonds and electrostatic interactions, the conformational flexibility is also limitedby energetic factors, as the motions of groups are likely to modify these interactions.Some of the conformational fluctuations of proteins are unmistakably functionallyimportant. For instance, the functioning of a protein can involve a structural change inducedby external stimuli or by thermal fluctuations. In fact, myoglobin is a good example of theneed for conformational flexibility. The protein can exist in two states, an oxygen-poor state(Mb), and an oxygen-rich state (MbO ). The functioning of the protein requires transitions2between these states. Dioxygen binds to the heme pocket of the protein, but within the nativestate the heme pocket is not exposed to the surroundings. Therefore, in order to function,structural fluctuations need to occur that allow small ligands to reversibly bind to the hemepocket. [Aus75] Consequently, if we wish to understand the design and functioning ofproteins it is not sufficient to just consider their tertiary conformations, but it is of equalimportance to study the fluctuations around their average structures. Conformational changesof proteins can also exert a negative influence on the functioning of biological systems. Forinstance, the prions that cause bovine sprongiform encephalopathy (BSE) and humanCreutzfeldt-Jakob disease are isoforms of normal cellular proteins that have adopted astructural change. [Pru97]In general, we can state that the functioning of a protein requires a delicate balancebetween order and disorder. Order can be found in the eedfor a well-defined structure,while disorder manifests itself in structural indeterminism and dynamics. These propertiesare reflected in the shape and structure of the potential ergy surface of a protein, oftenreferred to as its energy landscape. In terms of the ‘aperiodic crystal’, i.e. assumingcomplete structural order, the tertiary structure of a protein should correspond to a minimumwithin a very steep well on its energy surface. However, the aforementioned disorderedproperties of proteins require their energy surfaces to be much shallower. Furthermore, theyexhibit a rugged structure similar to the energy surfaces of disordered materials like glassesand polymers. [Fra91] More detailed information on the structure of protein nergylandscapes will be provided in the next chapter.1.2 Structural dynamics and spectral diffusionRecently, optical ine narrowing techniques such as hole burning and photon echospectroscopy have proven to be instrumental in elucidating the low-temperature dynamicEXPLORING PROTEIN ENERGY LANDSCAPES4properties of glasses. [Nar90, Mei94a, Mei94b, Koe96] The applicability of thesetechniques to the study of dynamics in low-temperature condensed phases relies on aphenomenon called spectral diffusion. The term spectral diffusion refers to the fluctuationsof the optical resonance frequency of a dye molecule due to structural fluctuations of thematrix it is dissolved in. With the use of photon echo and hole burning spectroscopy suchfluctuations can be studied over an extremely broad time window, ranging from picosecondsto several days. In this way, crucial information on the dynamic properties of the matrix canbe obtained. Apart from their extremely broad ynamic range, optical line narrowingtechniques are an extremely sensitive probe for structural dynamics, as their resolution isonly limited by the homogeneous linewidth, which, at liquid helium temperatures, can be assmall as a few thousandths of wavenumbers. The techniques are applicable to anymatrixthat can be doped with an optical probe. The fact that heme proteins, such as myoglobin andcytochrome c, contain an optical center, the heme group, as an intrinsic part of theirstructure, makes them particularly suitable for optical studies. Some preliminary experimentshave clearly demonstrated the general applicability of optical line narrowing techniques toheme proteins, [Box87, Kol87, Zol91a, Sai92] and have opened the way for an extensivestudy of their low-temperature dynamics and energy landscapes.1.3 Complex systemsApart from their biological significance, the dynamic behavior of biological systems isreceiving a rapidly increasing interest from workers in the physical sciences. This tendencyshould be viewed in the light of a shifting interest from ‘simple’ to ‘complex’ systems. Asit turns out, it is not always easy to distinguish simplicity from complexity. However, anillustration of some borderline cases may show that the study of complex systems calls fora different scientific approach. Physical or chemical systems may be characterized as simplebecause of their low dimensionality, such as atoms and small molecules, an inherentsymmetry, like that is present in perfect crystals, or the absence of interactions amongparticles in ideal gasses. A common motive in the treatment of simple systems is that anattempt is made to describe and understand the system in terms of the prop rties of theirbuilding blocks. For instance, the structure and dynamics of small molecules are treated interms of the properties of and the interactions between the atoms they are made up of. Forcomplex systems, such an approach is certainly not feasible, and may even prove to beobsolete. To give an extreme example, one cannot understand the behavior of a human beingby solving the Schrödinger equation for his brain, nor is it helpful to predict the trend of theglobal economy by considering each human factor individually. This does not mean that weare unable to study these systems, it just means that a different approach is required. In thesame way, physicists are able to study complex states of matter, including glasses, spinglasses, polymers, colloids, and biological systems. An important aspect of the study ofcomplexity is that systems of a wildly different nature, may share common properties, suchGENERAL INTRODUCTION5as hierarchical organization, self-similarity, self-organization and hysteresis. Defining suchproperties, and finding their possible common origin is of major importance to understandcomplex behavior. This is also true on an interpretational level, as is illustrated by thegeneral applicability of the landscape paradigms that are being used in this thesis. [Lan96]The same concepts can, for instance, be applied in tackling the traveling salesman problem,[Buc96] or in obtaining insight into the process of biological evolution.One thing that the study of simple and complex systems have in common is the need forwell defined model systems. Like atomic physics started with the simplest of atoms, thehydrogen atom, the natural course in the study of complex systems is to tart ut withcharacterizing the ‘simplest’ complex systems, and from that point on tackling morechallenging systems. As it turns out, many biological systems, including proteins, are idealmodel systems. One reason for this is that the structures of many biological systems are wellknown, which both facilitates both computational approaches, and the interpretation ofexperiments. This certainly holds for proteins, which have a number of additionaladvantages. They are often easily accessible, while their relatively modest size makes itpossible to prepare and study large ensembles. Proteins should be of particular interest tothose physicists involved with structurally disordered systems, like glasses and polymers.The dynamics of proteins are often described as being ‘glass-like’, which originates fromsimilarities between their potential energy surfaces. One of the goals of this thesis is to findhow far this analogy extends, and to explain their differences in terms of their energylandscapes.1.4 Scope and organization of this thesisThe aim of this thesis is to study low-temperature protein dynamics by performingspectral diffusion experiments on heme proteins. Secondly, an attempt will be made toconstruct an image of the protein’s energy landscape based on the results of theseexperiments. Furthermore, the protein-glass analogy will be critically evaluated.The main part of this thesis is written in such a way to be accessible to an audience thatis as wide as possible, and requires little background information, either for those interestedin protein structure and dynamics, or those with a general interest in spectral diffusionphenomena in disordered materials. Chapters 2 and 3 provide an introduction into the concepts and methods that will beapplied in the remainder of this thesis. These include the protein energy landscape andexisting models for its description, as well as experimental methods, and a quantitativemodel for the study of spectral diffusion. Experimental details are presented in chapter 4. Inchapter 5 we will present the results of spectral diffusion experiments on Zn-substitutedmyoglobin and cytochrome c. Based on these results, and the recent results of other groups,a highly detailed description of the energy landscape of myoglobin will be presented. Finally,in chapter 6, we will reflect on ultrafast protein dynamics at low temperature, by studyingEXPLORING PROTEIN ENERGY LANDSCAPES6the pure dephasing in Zn-myoglobin. GENERAL INTRODUCTION7

Exploring protein energy landscapes

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Exploring protein energy landscapes

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