Functional characterization of dolichol phosphate mannose synthases and development of infrared nanoscopy to study membrane proteins in solution
Time: Tue 2026-01-27 13.00
Location: Kollegiesalen, Brinellvägen 8, Stockholm
Video link: https://kth-se.zoom.us/j/61935309457
Language: English
Subject area: Biotechnology
Doctoral student: Markus M. Keskitalo , Industriell bioteknologi
Opponent: Professor Janne Ihalainen, University of Jyväskylä
Supervisor: Professor Christina Divne, Industriell bioteknologi; Professor C. Magnus Johnson, Yt- och korrosionsvetenskap
QC 2025-12-12
Abstract
Membrane proteins are proteins that are embedded in the lipid bilayers oforganisms. Roughly a fourth of all human proteins are estimated to bemembrane proteins and about 60 % of human-approved medications targetmembrane proteins. The correct function of membrane proteins is essential toall organisms.
This thesis is made up of two parts. First, the biochemistry and function ofdolichol phosphate mannose synthases (DPMS) are investigated. Theseenzymes are responsible for the transfer of mannose from a nucleotide sugardonor to the acceptor lipid dolichol phosphate, forming dolichol phosphatemannose (Dol-P-Man). In eukaryotes and archaea, Dol-P-Man is the keymannose donor for mannosylation reactions inside the endoplasmic reticulum(ER) lumen or on the extracellular leaflet of cell membrane, respectively. Asthe synthesis of Dol-P-Man is known to take place on the cytoplasmic side ofthe ER membrane in eukaryotes or the cell membrane in archaea, the questionremains how Dol-P-Man is transported onto the other side of the membraneto serve as a mannose donor. This thesis presents a hypothesis in which theDPMS itself is responsible for the flipping of its own product. The hypothesisis supported by crystallographic data that shows Dol-P-Man bound to a DPMSin a “flipped” orientation that could enable the transport to the other side ofthe membrane. This thesis also covers the recombinant expression,purification, and in vitro characterization of DPMS from the zebra fish Daniorerio. This DPMS is similar to the human enzyme and can therefore yieldmechanistic details behind DPMS-related diseases.
The second part covers the development of scattering-type scanning near-fieldoptical microscopy (s-SNOM) to study proteins in solution. The method iscapable of collecting images and infrared spectra from samples at nanometerscalelateral resolution. The method is not readily applicable for the study ofobjects in solution, but this limitation can be circumvented by the use of aliquid cell. The liquid cell is first used to probe the stretching vibrations ofwater in nanoscale and the method is then further developed and is applied tocollect images and spectra from purple membranes, a model membranecomprising tightly packed bacteriorhodopsin molecules and associated lipids.