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Biophysical studies of the membrane location of the voltage-gated sensors in HsapBK and KvAP
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
(English)Manuscript (Other academic)
Identifiers
URN: urn:nbn:se:su:diva-24834OAI: oai:DiVA.org:su-24834DiVA: diva2:198396
Note
Part of urn:nbn:se:su:diva-7488Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2010-01-14Bibliographically approved
In thesis
1. Structure and Dynamics of Membrane Associated Peptides
Open this publication in new window or tab >>Structure and Dynamics of Membrane Associated Peptides
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The peptide-membrane interaction is a key element for many biological functions, from cell signaling to cell internalization. In this thesis the peptide-membrane interaction of six different peptides have been studied with respect to their structure, membrane location and dynamics with spectroscopic methods. Penetratin and the N-terminal sequence of the bovine prion protein (1-30), bPrPp, belong to a class of peptides called cell-penetrating peptides (CPPs). CPPs are short, often highly basic peptides that have the ability to facilitate translocation of an attached hydrophilic cargo over cell-membrane. CD and NMR spectroscopy reveled that penetratin, the (supposedly) non-penetrating mutant pentratin(W48F,W56F) and bPrPp are all highly helical in membrane mimicking media. The position with respect to the bilayer is, however, very different for the three peptides, Penetratin is residing on the membrane surface with a slight tilt while bPrPp is transmembrane and penetratin(W48F,W56F) is somewere in between. These differences can explain the different impact these peptides have on membranes

We have also shown that penetratin can escape from vesicles when an electrochemical or pH gradient is present over the membrane, which support endocytotic internalization.

Melittin is a 26 amino acid long residue long peptide and is the major component of the European honey bee venom. Many studies have shown that melittin induces a transient pore that causes leakage in both natural and artificial membranes. In paper IV we used melittin as a model-peptide to investigate how peptides affect lipid dynamics in model-membranes. We showed that carbon-13 relaxation of the lipids could be used to characterize peptide induced changes in lipid dynamics

The voltage sensor is a domain of the voltage-dependent potassium channel containing several positively charged amino acids (usually arginines). The sensor undergoes a conformational change as a response to a membrane potential. Here, we have studied the membrane location of two fragments corresponding to the “paddle” domain of two different potassium channels, KvAP and HsapBK. NMR and fluorescence studies indicate that both peptides reside inside of the hydrophobic interior of the bilayer, which show that the fragment behave the same way as it does in the intact protein.

All six of these peptides interact strongly with model-membranes and adopt a helical conformation even though they have very different biological function. The difference in biological function can instead be explained by the variation in membrane position and membrane dynamics of these peptides

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 51 p.
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-7488 (URN)978-91-7155-641-7 (ISBN)
Public defence
2008-05-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-05-08 Created: 2008-05-08Bibliographically approved
2. Biophysical studies of membrane associated peptides
Open this publication in new window or tab >>Biophysical studies of membrane associated peptides
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A large part of the processes in living organisms involves proteins acting in a biological membrane. Biophysical studies on isolated model systems can give important understandings of the complicated biological mechanisms in the membrane. In this thesis peptide membrane interaction mechanisms are studied in several different systems. The membrane interactions of the unstructured endogenous opioid peptides dynorphinA (DynA) and dynorphinB (DynB) were investigated with Saturation Transfer Difference (STD) experiments, supplemented by various other NMR methods. The combined results support a conclusion that DynA binds to the lipid bilayer with the N-terminal residues inserted into the hydrophobic region and the C-terminal residues more loosely attached to the surface, while DynB is situated parallel to the bilayer. This difference in membrane interaction can explain observations that DynA has membrane perturbing effects while DynB has not. In the second study the binding domain of the glycosyltransferase A.laidlawii Monoglycosyldiacyl Glycerol Synthase (alMGS) was predicted and investigated mainly with NMR which enabled the determination of the 3D structure and position in a lipid environment. The phospholipid bilayers induce a large amphipathic α-helical content in the peptide, which aligns parallel but slightly tilted along the lipid surface with the N-terminus situated closer to the hydrophobic region. Lipid perturbation effects caused by peptide-membrane interactions were investigated by studying the influence of model transmembrane peptides on lipid dynamics in phospholipid bicelles with varying bilayer thickness. 13C-relaxation NMR of the lipids was used to survey the effects of the model peptides on the lipid bilayer.In paper IV and V structure and membrane interaction properties of the highly charged and flexible helix-turn-helix motif named the 'voltage sensor paddle' from two transmebrane voltage gated potassium channels was investigated. In membrane mimetic media, the KvaP paddle adopts the same type of helix-turn-helix conformation which can be seen in the Xray structure of the entire ion channel. The membrane interaction of the paddle HsapBK was compared with the corresponding one in KvaP, and both were inserted in the lipid bilayer but perturbed the lipid system differently, which may indicate differences in their function. Paper VI treats the structure of the novel site-specific fluorophore ReAsH bound to an optimized peptide sequence. The analysis shows that the important peptide mid segment configuration of CCPGCC is optimal for the ReAsH binding and that the N-terminal Phe1 plays an important role for the fluorophore process.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2009. 55 p.
Keyword
NMR, spectroscopy, membrane interaction, bicelles
National Category
Chemical Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-27488 (URN)978-91-7155-888-6 (ISBN)
Public defence
2009-06-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2009-05-14 Created: 2009-05-05 Last updated: 2012-08-28Bibliographically approved

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