Functional characterization of dolichol phosphate mannose synthases and development of infrared nanoscopy to study membrane proteins in solution

QC 2025-12-12

Abstract

Membrane proteins are proteins that are embedded in the lipid bilayers of

organisms. Roughly a fourth of all human proteins are estimated to be

membrane proteins and about 60 % of human-approved medications target

membrane proteins. The correct function of membrane proteins is essential to

all organisms.

This thesis is made up of two parts. First, the biochemistry and function of

dolichol phosphate mannose synthases (DPMS) are investigated. These

enzymes are responsible for the transfer of mannose from a nucleotide sugar

donor to the acceptor lipid dolichol phosphate, forming dolichol phosphate

mannose (Dol-P-Man). In eukaryotes and archaea, Dol-P-Man is the key

mannose donor for mannosylation reactions inside the endoplasmic reticulum

(ER) lumen or on the extracellular leaflet of cell membrane, respectively. As

the synthesis of Dol-P-Man is known to take place on the cytoplasmic side of

the ER membrane in eukaryotes or the cell membrane in archaea, the question

remains how Dol-P-Man is transported onto the other side of the membrane

to serve as a mannose donor. This thesis presents a hypothesis in which the

DPMS itself is responsible for the flipping of its own product. The hypothesis

is supported by crystallographic data that shows Dol-P-Man bound to a DPMS

in a “flipped” orientation that could enable the transport to the other side of

the membrane. This thesis also covers the recombinant expression,

purification, and in vitro characterization of DPMS from the zebra fish Danio

rerio. This DPMS is similar to the human enzyme and can therefore yield

mechanistic details behind DPMS-related diseases.

The second part covers the development of scattering-type scanning near-field

optical microscopy (s-SNOM) to study proteins in solution. The method is

capable of collecting images and infrared spectra from samples at nanometerscale

lateral resolution. The method is not readily applicable for the study of

objects in solution, but this limitation can be circumvented by the use of a

liquid cell. The liquid cell is first used to probe the stretching vibrations of

water in nanoscale and the method is then further developed and is applied to

collect images and spectra from purple membranes, a model membrane

comprising tightly packed bacteriorhodopsin molecules and associated lipids.

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