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Solution structure of the HsapBK K+ channel voltage-sensor paddle sequence
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.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 25, 5813-5821 p.Article in journal (Refereed) Published
Abstract [en]

Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.

Place, publisher, year, edition, pages
2009. Vol. 48, no 25, 5813-5821 p.
Keyword [en]
NMR solution structure, S3b−S4 fragment, paddle
National Category
Biophysics
Research subject
Biophysics; Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-31727DOI: 10.1021/bi9004599ISI: 000267326500006PubMedID: 19456106OAI: oai:DiVA.org:su-31727DiVA: diva2:278393
Funder
Swedish Research Council, 621-2011-5964
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2013-04-29
In thesis
1. NMR Investigations of Peptide-Membrane Interactions, Modulation of Peptide-Lipid Interaction as a Switch in Signaling across the Lipid Bilayer
Open this publication in new window or tab >>NMR Investigations of Peptide-Membrane Interactions, Modulation of Peptide-Lipid Interaction as a Switch in Signaling across the Lipid Bilayer
2010 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The complexity of multi cellular organisms demands systems that facilitate communicationbetween cells. The neurons in our brains for instance are specialized in this cell-cellcommunication. The flow of ions, through their different ion channels, across the membrane, isresponsible for almost all of the communication between neurons in the brain by changing theneurons membrane potentials. Voltage-gated ion channels open when a certain thresholdpotential is reached. This change in membrane potential is detected by voltage-sensors in the ionchannels. In this licentiate thesis the Homo sapiens voltage- and calcium-gated BK potassiumchannel (HsapBK) has been studied. The NMR solution structure of the voltage-sensor ofHsapBK was solved to shed light upon the voltage-gating in these channels. Structures of othervoltage-gated potassium channels (Kv) have been determined by other groups, enablingcomparison among different types of Kv channels. Interestingly, the peptide-lipid interactions ofthe voltage-sensor in HsapBK are crucial for its mechanism of action.Uni cellular organisms need to sense their environment too, to be able to move towardsmore favorable areas and from less favorable ones, and to adapt their gene profiles to currentcircumstances. This is accomplished by the two-component system, comprising a sensor proteinand a response regulator. The sensor protein transfers signals across the membrane to thecytoplasm. Many sensor proteins contain a HAMP domain close to the membrane that isinvolved in transmitting the signal. The mechanism of this transfer is not yet revealed. Ourstudies show that HAMP domains can be divided into two groups based on the membraneinteraction of their AS1 segments. Further, these two groups are suggested to work by differentmechanisms; one membrane-dependent and one membrane-independent mechanism.Both the voltage-gating mechanism and the signal transduction carried out by HAMPdomains in the membrane-dependent group, demand peptide-lipid interactions that can be readilymodulated. This modulation enables movement of peptides within membranes or within thelipid-water interface. These conditions make these peptides especially suitable for NMR studies.

Place, publisher, year, edition, pages
DBB, SU, 2010. 59 p.
Keyword
peptide-lipid interaction, membrane mimetic, NMR, biophysics
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-59534 (URN)
Presentation
2010-04-22, K205, Arrhenius Laboratories for Natural Sciences, Stockholm, 15:00 (English)
Opponent
Supervisors
Available from: 2012-01-25 Created: 2011-07-04 Last updated: 2012-01-25Bibliographically approved
2. Membrane Induced Structure in Transmembrane Signaling Proteins and Peptides: Peptide–Lipid Interactions Studied by Spectroscopic Methods
Open this publication in new window or tab >>Membrane Induced Structure in Transmembrane Signaling Proteins and Peptides: Peptide–Lipid Interactions Studied by Spectroscopic Methods
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological membranes, defining the boundary of cells and eukaryotic organelles, are mainly composed of lipids and membrane proteins. Interactions between these lipids and proteins are needed to preserve the tight seal of the membrane, but also to induce structure for proper function in many membrane proteins. In this thesis, interactions between three different kinds of peptides, i.e. small proteins, and model membranes are studied by spectroscopic methods.

First, the membrane interaction of two paddle domains, KvAPp, from the voltage-gated potassium channel KvAP from Aeropyrum pernix, and HsapBKp, from the human, large conductance, calcium-activated potassium channel HsapBK, was studied (paper I and II). In paper I, a high-resolution solution NMR structure of HsapBKp in detergent micelles is presented revealing a helix-turn-helix motif. Small structural differences between HsapBKp and KvAPp, positioning the arginines differently, are presented. These structural differences may explain why BK channels are weakly voltage-gated. In paper II, it is shown that HsapBKp perturbs the membrane more than KvAPp and that the membrane perturbation is related to β-structure and to dynamics in the turn in the helix-turn-helix motif.

Second, the membrane interaction of HAMP domains modulating transmission in prokaryotic transmembrane signaling was studied (paper III). Based on the membrane interaction of the AS1 segments of the HAMP domains, two groups were identified: one strongly membrane interacting and one weakly membrane interacting. The two groups are suggested to use different signaling mechanisms.

Third, nonspecific binding of proinsulin C-peptide, the linker peptide connecting chain A and B in insulin, to model membranes was studied (paper IV). The study revealed that C-peptide binds to a model membrane at low pH, but the membrane induces no large structural rearrangements of the peptide. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. 59 p.
Keyword
peptide-lipid interaction, paddle domain, voltage gating, voltage sensor domain, micelle, phospholipid bicelle, solution structure, HAMP domain, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, nonspecific interaction
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-79051 (URN)978-91-7447-564-7 (ISBN)
Public defence
2012-09-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2012-09-06 Created: 2012-08-24 Last updated: 2012-08-29Bibliographically approved
3. 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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