Direccionamiento Estratégico Modelo de Gestión

Nuclear magnetic resonance (NMR) spectroscopy has proved itself a potentially powerful method for the determination o f membrane protein three-dimensional structure.

Solid state NM R can be used to study an intact membrane protein by the determination o f intemuclear distances and orientations. The deconvolution o f the NMR spectra is usually performed by using specifically isotopically labelled proteins in combination either with the orientation o f the protein in the magnetic field, or with the sample rapidly rotating in the magnetic field (so-called magic angle spinning). These methods have been used to acquire structural information for membrane proteins [Wang et al., 1997; Grobner et al., 1998; Kumashiro et al., 1998; W illiamson et al., 1998] and

Chapter 1. Introduction

membrane protein fragments [Bechinger et al., 1993; Ketchem et al., 1997; Rigby et al., 1998].

High resolution solution NMR spectroscopic methods have been used in this work, and therefore w ill be described in more detail. There are numerous texts that describe this methods and its applications [for example, Derome, 1987]

Each o f the four most abundant elements in biological material (H, C, N, and O) have at least one naturally occurring isotope with non-zero nuclear spin and is in principle observable in an NM R experiment. The naturally occurring isotope o f hydrogen, *H, is present at >99% abundance and forms the basis o f the experiments described here. Other important NMR-active isotopes include '^C and *^N present at 1.1 and 0.4% natural abundance, respectively. The low natural abundance o f these two isotopes makes their observation difficult on commonly isolated natural products. These two nuclei are however very extensively used for larger (>10 kD) proteins which can be isotopically enriched (to >95% if necessary) when cloned into over expression systems.

The spin angular momentum o f nuclei with isotopes o f overall non-zero spin w ill undergo a rotation motion called precession in the presence o f an external magnetic field. The frequency o f precession for each isotope is dependent on the strength o f the external field and is unique for each isotope. For example, in a magnetic field o f a given strength (e.g. 14.1 Tesla) all protons in a molecule w ill have characteristic resonance frequencies (chemical shifts) within a dozen or so parts per m illion (ppm) o f a constant value (e.g., 600.13 MHz) characteristic o f the particular nuclear type. These slight differences are due to the type o f atom the proton is bound (e.g., C, N, O, or S), the local chemical environment and conformation o f the molecule. Thus each proton should, in principle, be characterized by a unique chemical shift. In practice, this is never observed as some protons are found

Chapter 1. Introduction

to have degenerate chemical shifts. Other protons (e.g., some OH, SH, and NH3) are in rapid chemical exchange with the solvent and thus have chemical shifts indistinguishable from the solvent resonance. However, complete chemical shift assignments are often possible and are a prerequisite for structural studies using NM R parameters.

Structural information from NM R experiments come primarily from through-bond (scalar or J coupling) or through space (the nuclear Overhauser effect, NOE) magnetization transfer between pairs o f protons. J couplings between pairs o f protons separated by three or fewer covalent bonds can be measured. The value o f a three-bond J coupling constant contains information about the intervening torsion angle, described by co called Karplus relationship:

= A cos (0) + 5 cos^ (0) + C Eq. 1.1

where A, B, and C are empirically derived constants for each type o f coupling constant (e.g., 3 J„hnh or 3 JaHpu)* Torsion angles cannot be unambiguously determined from a Karplus-type relationship since as many as four different torsion angle values correlate with a single coupling constant value. However, constraints on the dihedral angles (|) are important structural parameters in the determination o f protein three-dimensional structures by NMR.

The other maj or source o f structural information comes from through space dipole- dipole coupling between two protons called the NOE. The intensity o f an NOE is proportional to the inverse o f the sixth power o f the distance separating the two protons and is usually observed if two protons are separated by less than 5 Â. The use o f the NOE intensities in the structural determination was pioneered by Gibbons and co-workers [Gibbons et al., 1975; Jones et al., 1978]. The NOE is a sensitive probe o f short intramolecular distances. NOEs are categorized according to the location o f the two protons

Chapter 1. Introduction

involved in the interaction. A network o f these short inter-proton distances form the backbone o f three-dimensional structure determination by ID, 2D and 3D NMR.

A number o f short distances are fairly unique to secondary structural elements. For example, a-helices are characterized by short distances between certain protons on sequentially neighboring residues (e.g., between backbone amide protons, d ^ , as w ell as between beta protons o f residue i and the amide protons o f residue i+1, dp^). Helical conformations result in short distances between the alpha proton o f residue i and the amide proton o f residues i+3 and to a lesser extent i+4 and i+2. These i+2, i+3, and i+4 NOEs are collectively referred to as medium range NOEs while NOEs connecting residues separated by more than 5 residues are referred to as long range. Extended conformations (e.g., P- strands) on the other hand, are characterized by short sequential, d^^, distances. The formation o f sheets also result in short distances between protons on adjacent strands (e.g.,

d a a and d^^).

The procedure for structure determination generally proceeds as follows: assign unequivocally all the molecule's resonances, measure the intensity o f all the NOEs observed and calculate structures that match the experimental data.

Before a structure can be determined the pairs o f protons responsible for each o f the NOEs observed must be identified. The only way to do this is to assign every resonance in the molecule's spectrum. The strategy for assigning protein resonances known as sequential assignment was first developed by Gibbons and coworkers [Gibbons et al., 1975 ; Jones et al., 1978a, 1978b; Kuo et al., 1979; Ford et al., 1979], followed by Wuthrich and coworkers [Wutrich et al, 1979, 1982].

The NMR-based structures could be produced by Distance Geometry [Havel and Wuthrich, 1984, 1985] and/or Restrained Molecular Dynamics, as described below.

C hapter 1. Introduction

The quality o f the NMR based protein structures depends on 1) the number o f NOE distance constraints, 2) their assumed precision, 3) the method o f structure calculation and 4) the size o f the protein. The influence o f these parameters was studied [Liu et al., 1992], and it was found that 1) global RMS decrease as the number o f constraints increases up to 30% o f o f all potential constraints; 2) the accuracy o f the average structure calculated by the Restrained Molecular Dynamics is greater than that structures obtained by purely geometric methods.