2D Nmr Studies of Biomolecules: Protein Structures and Protein-Dna Interactions 2D Nmr Studies of Biomolecules: Protein Structures and Protein-Dna Interactions
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2D NMR STUDIES OF BIOMOLECULES: PROTEIN STRUCTURES AND PROTEIN-DNA INTERACTIONS 2D NMR STUDIES OF BIOMOLECULES: PROTEIN STRUCTURES AND PROTEIN-DNA INTERACTIONS 2D NMR STUDIES VAN BIOMOLECULEN: EIWITSTRUCTUREN EN EIWIT-DNA INTERACTIES (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Rijksuniversiteit te Utrecht op gezag van de Rector Magnificus, Prof. Dr. J.A van Ginkel ingevolge het besluit van het College van Dekanen in het openbaar te verdedigen op 25 oktober 1989 des namiddags te 12.45 uur door Rudolf Mat hias Johannes Nicolaas Lamerichs geboren op 1 oktober 1959 te Echt Promotor: Prof. Dr. R. Kaptein Co-promotor: Dr. R. Boelens aan mijn ouders ter herinnering aan mijn vader Voorwoord Naarmate de kennis over de wereld om ons heen toeneemt, lijkt het erop dat onderzoek steeds meer "team-work" wordt. Meer en meer mensen werken, ieder binnen zijn of haar eigen specialisme, aan een bepaald onderwerp. Dit geldt ook voor het onderzoek beschreven in dit proefschrift. Allereerst ben ik dank verschuldigd aan mijn promotor, Rob Kaptein, die mij gedurende de afgelopen vier jaar ruime mogelijkheden geboden heeft om het werk te verrichten dat geresulteerd heeft in dit proefschrift. Mijn co-promotor, Rolf Boelens, was onontbeerlijk om mij regelmatig weer op het goede spoor te zetten. Ton Ruilmann en André Padilla hebben mij wegwijs gemaakt in de jungle van programmatuur die tegenwoordig beschikbaar is om molekuulstructuren te bepalen. Verder wil ik alle collega's hartelijk danken voor alle bijdragen die zij geleverd hebben. Het enthousiasme van Larry Berliner mag daarbij zeker niet ongenoemd blijven. Het onderzoek is tegenwoordig niet alleen "team-work" geworden, ook komt het steeds vaker voor dat samenwerking met andere groepen noodzakelijk is, met name om het materiaal waar aan gemeten wordt te verkrijgen. In dit verband ben ik dank verschuldigd aan Miguel Llinâs, Brian Fera, Gijs van der Marel en Manfred Schnarr. Tenslotte wil ik Jan den Boesterd bedanken voor het uitstekende fotowerk. Contents 1 Introduction. 3 2 Secondary structure of crambin in solution. 25 A two-dimensional NMR study. 3 NMR study of the solution structure of crambin. 39 Sequential and stereospecific assignment of the proton resonances anu structure determination using an iterative relaxation matrix approach. 4 1H NMR study of a complex between lac repressor headpiece 83 and a 22 base pair symmetric lac operator. 5 Assiqnment of the proton NMR spectrum of a lac repressor 101 headpiece-operator complex in H2O and identification of NOEs: consequences for the model of protein-DNA interaction. 6 The amino-terminal domain of LexA repressor is a helical 129 but differs from the canonical helix-turn-helix proteins: A two dimensional 1H NMR study. Summary 145 Samenvatting 147 Curriculum vitae 151 Chapter 1 Introduction For a long time X-ray crystallography was the only technique available for determining the three dimensional structure of proteins. The method has been highly successful and by now over 300 protein structures have been solved. Nevertheless, a limitation of the method is that proteins must be available in crystalline form. Of more recent date is the notion that nuclear magnetic resonance (NMR) can provide information from which the solution structures of biomolecules can be derived. Using NMR it is possible to measure the distance between two nuclei. The dipole-dipole interaction between them will result in magnetisation transfer, i.e. the nuclear Overhauser effect (NOE). Since the NOE can be measured in solution, no crystals are needed. A prerequisite for structure determination is that most resonances have been assigned. The development of the two-dimensional (2D) NMR techiques made it possible to assign most proton resonances for proteins with a molecular weight smaller than ca. 15 kDa. Also the availability of stable high-field magnets was essential for the results obtained by NMR over the last decade. This thesis decribes the use of NMR to derive high resolution structures of proteins in solution (chapters 2 and 3) and the application of NMR to a problem in molecular biology, i.e the specific recognition of DNA sequences by proteins (chapters 5-6). In the following a short introduction to these subjects will be given. BIOMOLECULAR STRUCTURES FROM NMR As already mentioned, the dipole-dipole interaction between protons is the basis for structure determination by NMR. In the 2D NOE spectra, this interaction results in a set of off-diagonal cross-peaks [1]. The intensity of these cross-peaks wili depend on the distance between the protons and the time during which the protons are allowed to interact with each other, the mixing time [1]. For instance, for longer mixing times (200-300 ms) a proton will show NOEs with all protons which are within a distance of ca. 5 A. The information present in 2D NOE spectra is used in two ways. First, the sequential NOEs between the backbone amide and Ca protons are used for the resonance assignment. Two short distances, between the Ca proton of residue i and the amide of i+1 (daN) and between the amide protons of neighbouring residues (dNN), give rise to these connectivities. Secondly, the NOEs contain information required for structure determination. The secondary structure is determined by medium range NOEs and the intensities of the daN and dNN connectivities [1], For instance, for an extended backbone conformation the daN connectivities are strong whereas the dNN connectivities are weak or absent. In helices the opposite applies and furthermore, the distance between the Ca proton of residue i and both the amide and CP protons of residues i+3 will be short enough to give NOEs, dnN (i,i+3) and dap (i,i+3) connectivities, respectively. The daN (i,i+4) connectivities are typical for a helices whereas 310 helices and tight turns show NOEs between residues i and i+2. The tertiary structure is predominantly determined by long range NOEs, i.e. those between protons of residues which are separated by more than four positions in the sequence. Therefore, NMR based structures rely on the availability of a consistent set of these long range NOEs. The next step is to translate these long range NOEs or distance constraints into actual 3D structures. In recent years several computational methods have been used for this purpose. The distance geometry (DG) algorithm [2-4] tries to fold the peptide backbone in such way that all distance constraints can be satisfied. Usually, this procedure does not yield one unique structure. Therefore, it is repeated several times until a family of related structures is obtained. The differences between the structures can be considered to be a measure of the resolution. To improve the sampling of the allowed conformational space for a given set of distance constraints one can use distance bounds driven dynamics (DDD) [5,6]. Starting from a structure, for instance obtained by distance geometry, Newtons equations of motion SV m. r. = (1) are solved. The energy function is the same as used in the distance geometry optimisation step. It contains the difference between the proton-proton distance in the input structure and the distance measured by NMR and a chiral error function. In this way the structure is 'shaken up'. The structures obtained in this way can be further refined taken into account an energy function with a true physical meaning. In restrained molecular dynamics MD [7,8] again Newtons equations of motion (Eq. 1) are solved. The energy function has the form: V = Vbo^ + Vang,e + Vdihr + VvdW + V^i^t, + Vdc. (2) Here, Vbond and Vang|e keep the bond lengths and angles close to their equilibrium values. Vdjhr describes rotation around bonds using a sinusoidal potential. VvdW is usually described by a Lennard-Jones potential and VCOU|Onnb takes electrostatic interactions into account. The last term, Vdc, is a pseudo potential that accounts for the distance constraints, in a similar way as described above for the DDD algorithm. Sofar the distance constraints have not been considered in detail. There are several ways to handle the NOE information derived from the spectra. They can be used in a qualitative manner: if an NOE is detected between two protons the distance between them must be shorter than 5 A [5]. To handle the information in a quantitative manner, the NOE intensity must be measured for several mixing times. In a first approximation, the coss-relaxation rate (a,j) is determined from the initial build-up rate and assumed to be proportional to the inverse sixth power of the distance (d,,) [1]: oij = kdi,-6 (3). In this way proton-proton distances can be obtained which can be used in the DG, DDD or restrained MD calculations. However, the NOE intensity can be affected by spin diffusion [9], In particular, if the protons are relatively far apart, the distance calculated according to Eq. 3 can be shorter that the real distance [10]. The contribution of spin diffusion can be calculated by so'ving the Bloch equations for the complete set of dipolar coupled protons [10,11]. An iterative relaxation matrix approach (IRMA) [12,13] has been proposed which takes into account the effect of spin diffusion on the NOE intensities. Based on a model structure for the biomolecule, improved proton-proton distances can be obtained which in turn can be used to derive a better structure. PROTEIN-DNA INTERACTION The helix-turn-helix motif. In the early eighties the X-ray structures of several DNA binding proteins were solved, i.e. the catabolite gene activating protein (CAP) [14], and the cl repressor (or >. repressor) [15] and cro repressor [16] of bacteriophage X. Both X repressor and cro control the life cycle of the phage by binding to specific operators on the DNA.