Structure and Dynamics of Coil-like Molecules by Residual Dipolar Couplings

Nuclear magnetic resonance (NMR) spectroscopy provides us with many means to study biological macromolecules in solution. Proteins in particular are the most intriguing targets for NMR studies. Protein functions are usually ascribed to specific three-dimensional structures but more recently tails, long loops and non-structural polypeptides have also been shown to be biologically active. Examples include prions, -synuclein, amylin and the NEF HIV-protein. However, conformational preferences in coil-like molecules are difficult to study by traditional methods. Residual dipolar couplings (RDCs) have opened up new opportunities; however their analysis is not trivial. Here we show how to interpret RDCs from these weakly structured molecules. The most notable residual dipolar couplings arise from steric obstruction effects. In dilute liquid crystalline media as well as in anisotropic gels polypeptides encounter nematogens. The shape of a polypeptide conformation limits the encounter with the nematogen. The most elongated conformations may come closest whereas the most compact remain furthest away. As a result there is slightly more room in the solution for the extended than for the compact conformations. This conformation-dependent concentration effect leads to a bias in the measured data. The measured values are not arithmetic averages but essentially weighted averages over conformations. The overall effect can be calculated for random flight chains and simulated for more realistic molecular models. Earlier there was an implicit thought that weakly structured or non-structural molecules would not yield to any observable residual dipolar couplings. However, in the pioneering study by Shortle and Ackerman RDCs were clearly observed. We repeated the study for urea-denatured protein at high temperature and also observed indisputably RDCs. This was very convincing to us but we could not possibly accept the proposed reason for the non-zero RDCs, namely that there would be some residual structure left in the protein that to our understanding was fully denatured. We proceeded to gain understanding via simulations and elementary experiments. In measurements we used simple homopolymers with only two labelled residues and we simulated the data to learn more about the origin of RDCs. We realized that RDCs depend on the position of the residue as well as on the length of the polypeptide. Investigations resulted in a theoretical model for RDCs from coil-like molecules. Later we extended the studies by molecular dynamics. Somewhat surprisingly the effects are small for non-structured molecules whereas the bias may be large for a small compact protein. All in all the work gave clear and unambiguous results on how to interpret RDCs as structural and dynamic parameters of weakly structured proteins.Ydinmagnettinen resonanssi spektroskopia (NMR) tarjoaa mahdollisuuden tutkia biologisia molekyylejä lioksessa. Varsinkin proteiinit ovat oleet tutkimusten kohteina. Proteiinien on perinteisesti ajateltu olevan aktiivisia vain laskostuneessa, kolmiuloittesessa muodossaan. Viimeaikoina on kuitenkin saatu yhä enemmän tietoa myös rakenteettomien tai heikosti rakenteellisten osien tärkeydestä proteiinien toiminnassa. Näiden osien tutkiminen perinteisin menetelmin on kuitenkin osoittautunu vaikeaksi. Jäännösdipolikytkentä (RDC) on tuonut uusia mahdollisuuksia tutkimuksiin. Jäännösdipolikytkentä tulee haivattavaksi kun käytetään ympäristöä, missä molekyyli törmäilee liuoksessa olevan esteen kanssa ja näin yleensä nollaan keskiarvoistuva kytkentä on havaittavissa. Molekyylin muodosta riippuu miten lähelle sen keskipiste pääsee tätä estettä tai seinämää. Tästä syystä johtuva virhe tulee ottaa huomioon tulosten tarkastelussa. Aikaisemmin luultiin, että laskostumattomista tai heikosti laskostuneista molekyyleistä ei voida mitata jäännösdipolikytkentöjä. Kuitenkin Shortle ja Ackerman ovat töissään osoittaneet, että tämä on mahdollista. Jatkoimme tutkimuksia käyttäen mallina homoplymeerejä ja jatkoimme molekyyli dynamiikkaan. Työmme tuloksena syntyi selvä kuva miten laskostumattomia molekyylejä tulisi mitata jäännösdipolikytkentöjen avulla