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.

Membranproteiner är proteiner som är inbäddade i organismers cell- ellerorganellmembran. Ungefär en fjärdedel av alla mänskliga proteineruppskattas vara membranproteiner, och cirka 60 % av alla läkemedel riktar sigmot membranproteiner. Korrekt fungerande membranproteiner är docklivsavgörande för alla levande organismer.

Denna avhandling består av två delar. Först undersöksdolikolfosfatmannossyntaser (DPMS) med avseende på biokemi och funktion.Dessa enzymer ansvarar för överföringen av mannos från ennukleotidsockerdonator till acceptorlipiden dolikolfosfat varviddolikolfosfatmannos (Dol-P-Man) bildas. I eukaryoter och arkéer är Dol-PManden viktigaste mannosdonatorn för mannosyleringsreaktioner i lumen avdet endoplasmatiska retiklet (ER), samt på den extracellulära sidan avcellmembranet. Man vet att syntes av Dol-P-Man sker på den cytoplasmatiskasidan av ER-membranet, motsvarande det yttre cellmembranet hos arkéer,men frågan kvarstår gällande hur Dol-P-Man transporteras till den andrasidan där den kan fungera som mannosdonator. Avhandlingen presenterar enhypotes där DPMS självt ansvarar för transport av den egna produkten.Hypotesen stöds av kristallografiskt data där Dol-P-Man observeras bundet tillDPMS i ”omvänd orientering” (flipped) som skulle kunna möjliggöra transporttill andra sidan av membranet. Avhandling omfattar även framgångsrikproduktion, rening och funktionell karakterisering av eukaryot DPMS frånmodellorganismen Danio rerio (zebrafisk). Denna typ av DPMS liknar detmänskliga enzymet och kan därför bidra med ökad förstålse för mekanistiskadetaljer bakom DPMS-relaterade sjukdomar.

Den andra delen av avhandlingen berör utveckling av spridningsbaseradnärfältsoptisk mikroskopi (s-SNOM) för att studera proteiner i lösning.Metoden kan generera bilder och infraröda spektra från prover till en lateralupplösning i nanometerområdet. Att använde s-SNOM för studier av objekt ilösning är inte möjligt med ett normalt provsteg men denna begränsning kankringgås med hjälp av en vätskeprovcell. Vätskeprovcellen används först föratt undersöka vattnets vibrerande töjning, och därefter utvecklas metodenvidare för datainsamling på modellmembran bestående av tätt packadebakteriorhodopsinmolekyler och tillhörande lipider (purple membranes).

Nano-FTIR spectroscopy is a technique where atomic force microscopy (AFM) and infrared (IR) spectroscopy are combined to obtain chemical information with a lateral resolution of some tens of nm. It has been used to study numerous solid surfaces and recently also liquids including water have been examined by separating the liquid from the AFM tip by a thin lid. However, although the water stretching vibrations are significantly more intense than the bending vibration in conventional IR spectroscopy, only the bending vibration has been observed in nano-FTIR spectroscopy so far. In this article we show that also the stretching vibrations of liquid H2O, D2O, HOD, and aqueous salt solutions can be probed in nano-FTIR spectroscopy. Nano-FTIR absorption and phase spectra have been acquired at different harmonics and it was found the third optical harmonic (O3) in the nano-FTIR absorption spectra exhibited the highest resemblance with attenuated total reflection (ATR). Being able to probe water stretching vibrations with nano-FTIR spectroscopy is of importance since the stretching vibrations contain considerably more detailed information regarding for example hydrogen bonding strength than the bending vibration. In addition, it enables studies of the essential interactions between water and biomolecules. Furthermore, this work highlights both advantages and challenges that nano-FTIR spectroscopy in a liquid sample cell has and importance of further studies that lead to a better understanding of the near-field signals in liquids.

The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-beta-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-beta-1,4-mannosidase [EC 3.2.1.25] that is highly specific for beta-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 angstrom resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family beta-galactosidases and the GH164-family exo-beta-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 beta-galactosidase-type homotrimeric structure.