In the field of bioscience there is an ongoing explosive growth in discovery and information. Novel means in biotechnology as well as in medicines are introduced at an unseen rate. One of the aspects contributing to this development is the increased understanding of protein function and structure. Proteins have a role in almost every biological process. The function and structure of proteins are linked. Recent studies have discovered that the understanding of the protein structure has been biased. Namely, the studies have unearthed a previously dismissed protein structure state: intrinsically disordered proteins (IDPs). In this highly dynamic state a protein is without a globular fold, but does not meet the requirements of a random coil either. Rapid transition between folds renders most of the established research techniques to be poor methods to study the IDPs.
Nuclear magnetic resonance (NMR) is a spectroscopy method, which enables the study of molecules at atomic resolution. The technique is based upon manipulation of the nuclear spins in specifically produced sample under strong magnetic field. In this method, spins of the system generate quantum coherence state(s), which is utilized to obtain information about the system. NMR is suitable for studying samples in solid and liquid mediums, but in case of biomolecules, water solution is preferable as it resembles in vivo environment. Highly mobile structure and chemical composition of IDPs cause many established NMR experiments to fail. Development of NMR pulse sequences is an obvious approach to solve the problem.
This thesis presents a number of NMR pulse sequences, which are designed to improve acquisition of information from highly mobile sections of proteins. The key aspect is to utilize H atom instead of HN in coherence transfer. Additional improvements include limited residue specific identification and novel coherence transfer pathways.
Articles I, II, and III present triple resonance experiments, which correlate protein backbone atoms. Combination of the spectra enables full sequential assignment. Article IV introduces an improved pulse sequence for measuring J couplings between nitrogen and amide proton.
The experiments were subjected to experimental verification. Comparisons were drawn between established pulse sequences. In both globular proteins and IDPs the results show improvement over established pulse sequences. The proposed sequences yielded improved assignment coverage, resolution and sensitivity enhancement.Biotieteiden alalla on käynnissä räjähdysmäinen kehitys. Uusia tutkimusmetodeja ja tuloksia julkaistaan ennennäkemättömällä tahdilla. Yksi tämän kehityksen mahdollistava tekijä on ymmärrys proteiinien rakenteesta ja toiminnasta. Proteiineilla on merkittävä rooli miltei kaikissa bio-prosesseissa. Proteiinien rakenteen muutoksilla on merkittävä rooli niiden toiminnassa. Viimeaikaiset tutkimukset ovat osoittaneet, että käsitys proteiinien rakenteesta on ollut puutteellinen. Rakenteellisen ja rakenteettomien tilojen välillä esiintyvä ns. intrinsically disordered proteiinit (IDP) esiintyvät dynaamisessa muodossa. Tämän tilan tutkiminen vakiintuneilla metodeilla on vaikeaa.
Ydinmagneettinen resonanssi (NMR) mahdollistaa molekyylien tutkimisen atomaarisella resoluutiolla. Tekniikka perustuu ydinspinin manipulaatioon voimakkaassa magneettikentässä. Spinit saatetaan kvanttikoherenssitilaan, joita observoimalla voidaan systeemistä saada tietoa. Metodi soveltuu dynaamisten systeemien tutkimiseen. Tämä tekee NMR:stä hyvän tekniikan IDP:n tutkimiseen. Kuitenkin vakiintuneet NMR:n metodit eivät suoraan sovellut IDP:n tutkimiseen. Väitöskirjassa esitellään muuteltuja ja paranneltuja NMR pulssisarjoja, jotka soveltuvat paremmin IDP:n tutkimiseen
