The B family of DNA-dependent DNA polymerases (pols) are uniquely sensitive to inhibition by N2-(p-n-butylphenyl)deoxyguanosine 5'-triphosphate (BuPdGTP). The affinity of members of the B family for BuPdGTP varies greatly, as does the ability of certain members to use the modified nucleotide as a substrate. For example, eukaryotic pol α has high affinity for the nucleotide, but incorporates it into DNA poorly, while T4 pol has lower affinity, but incorporates the nucleotide well. This thesis addresses two questions: 1) What are the amino acid residue(s) that impart sensitivity to BuPdGTP? and 2) is incorporation of BuPdGTP required for inhibition? To answer the first question, molecular modeling was used with the crystal structure of RB69 pol [Wang et al., 1997], an enzyme closely related to T4 pol [Wang et al., 1995]. This modeling identified a structural pocket adjacent to the polymerase active site that could serve as the butylphenyl "receptor". Based upon this modeling, a chimeric T4 pol containing the residues corresponding to the butylphenyl receptor from human pol α was designed and engineered for expression. The chimera was hypothesized to have a pol α-like phenotype with respect to its response to BuPdGTP (higher sensitivity/ lost ability to incorporate). The chimera was found to be unstable during purification, leaving the hypothesis unresolved. To answer the second question, non-substrate derivatives of BuPdGTP were in which the α,β anhydride oxygen of the triphosphate were replaced with either a CH2 or NH. The ability of the latter derivatives to inhibit polymerase activity and to serve as substrates was measured on T4 pol, RB69 pol and human pol α. Both derivatives retained high potency, but were not substrates under the conditions tested. These compounds were potent, selective inhibitors of B family pol that should be useful in the formation of a stable complex of enzyme:DNA:inhibitor for crystallization trials.

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The three-dimensional structure of a protein, formed as a result of amino-acid sequences folding into compact domains, is regarded as a key factor in its biological function. How and why proteins fold into specific topologies, remain the key focus of scientific research in the field of biophysics. By stripping down complex reactions down to the most basic elements, biophysicists aim to develop simplified models for biological phenomena such as antibody discrimination, viral fusion or self-assembly. Focusing on small model peptide systems, rather than the full proteins from which they were derived, was hoped to result in accurate structural measurements and provide a more transparent comparison between simulation and experiment. The aim of this research was therefore to investigate how accurate these models were when compared against experiment. Furthermore, while breaking down the complex biological phenomena into simple models, there was also a conscious effort to ensure that the models were representative of real biological systems, and a major focus was therefore aimed at determining whether any meaningful biomedical insight may be extrapolated from such models. Peptides found in hormones (human chorionic gonadotropin, luteinizing hormone), viruses (HIV) and amyloid diseases (transthyretin) were selected in order to probe a variety of questions in relation to the aforementioned biological phenomena. Namely, how the primary sequence influenced the three-dimensional structure (and thus its biological function), how its environment could influence such a confirmation, and how these systems aggregated. This doctoral study has made use of a combination of computer simulations and experimental techniques to investigate a selection of biologically relevant peptides; utilising classical atomistic molecular dynamics (MD) simulations to characterise the free-energy landscapes of the chosen peptides, and compare these findings with the secondary structure content predicted by spectroscopic methods such as circular dichroism and infrared spectroscopy. The peptide systems studied within, were found to be characterised by rugged free-energy landscapes unlike their protein counterparts (defined by singular, deep minima). Furthermore, these landscapes were found to be highly plastic and sensitive to changes in the local environment.

Torpedo AChR structure was used to make models of other LGICs. Coarse-grain MD allowed the identification of residues in the TM domain interacting with the lipid-bilayer. Born energy profiles through LGIC pores reveal that the EC domain plays a key role in ion selectivity.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 7 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 7 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.