Interresidual Distance Determination by Four-Pulse Double Electron-Electron Resonance in an Integral Membrane Protein: The Na+/Proline Transporter PutP of Escherichia coli

Jeschke, Gunnar; Wegener, Christoph; Nietschke, Monika; Jung, Heinrich; Steinhoff, Heinz‐Jürgen

doi:10.1016/s0006-3495(04)74310-6

Cited by 78 publications

(68 citation statements)

References 34 publications

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“…Around the same time a number of early applications demonstrated the feasibility of DEER and double-quantum EPR studies on spin-labeled peptides (14), soluble proteins (15,16), a pair of tyrosyl radicals in ribonucleotide reductase (RNR) (17), and integral membrane proteins (18). For the N-terminal domain of major plant light harvesting complex II (LHCII), which was missing in crystal structures, information on the conformational distribution could be obtained (19).…”

Section: Introductionmentioning

confidence: 99%

DEER Distance Measurements on Proteins

Jeschke

2012

Annu. Rev. Phys. Chem.

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Distance distributions between paramagnetic centers in the range from 1.8 to 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins can be measured by the DEER technique. The number of paramagnetic centers and their relative orientation can be characterized. DEER does not require crystallization and is not limited with respect to the size of the protein or protein complex. Diamagnetic proteins are accessible by site-directed spin labeling. To characterize structure or structural changes, the experimental protocols were optimized and techniques for artifact suppression were introduced. Data analysis programs were developed and it was realized that interpretation of the distance distributions must take into account the conformational distribution of spin labels. First methods have appeared for deriving structural models from a small number of distance constraints. The current scope and limitations of the technique are illustrated.

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Section: Introductionmentioning

confidence: 99%

DEER Distance Measurements on Proteins

Jeschke

2012

Annu. Rev. Phys. Chem.

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923

1,073

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“…Nonetheless, site-directed alkylation studies (compiled in ref. 4) clearly indicate that an outward-facing conformation with a hydrophilic pathway on the periplasmic side of LacY exists during turnover.Four-pulse double electron-electron resonance (DEER) (12, 13), combined with site-directed spin labeling (14,15), is well suited for distance measurements in proteins in the 20-to 60-Å range, which is comparable with the size of LacY, and the method has been applied successfully to membrane proteins (16)(17)(18)(19). The small size of the nitroxide spin-label minimizes uncertainty due to mobility of the label itself, and importantly, the signal reflects intramolecular interactions between two identical spin labels.…”

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Sugar binding induces an outward facing conformation of LacY

Смирнова

Kasho

Choe

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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According to x-ray structure, the lactose permease (LacY) is a monomer organized into N-and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single sugar-binding site at the apex. The periplasmic side of the molecule is closed. During sugar/H ؉ symport, a cavity facing the periplasmic side is thought to open with closure of the inwardfacing cytoplasmic cavity so that the sugar-binding site is alternately accessible to either face of the membrane. Double electronelectron resonance (DEER) is used here to measure interhelical distance changes induced by sugar binding to LacY. Nitroxidelabeled paired-Cys replacements were constructed at the ends of transmembrane helices on the cytoplasmic or periplasmic sides of wild-type LacY and in the conformationally restricted mutant Cys-1543 Gly. Distances were then determined in the presence of galactosidic or nongalactosidic sugars. Strikingly, specific binding causes conformational rearrangement on both sides of the molecule. On the cytoplasmic side, each of six nitroxide-labeled pairs exhibits decreased interspin distances ranging from 4 to 21 Å. Conversely, on the periplasmic side, each of three spin-labeled pairs shows increased distances ranging from 4 to 14 Å. Thus, the inward-facing cytoplasmic cavity closes, and a cleft opens on the tightly packed periplasmic side. In the Cys-1543 Gly mutant, sugarinduced closing is observed on the cytoplasmic face, but little or no change occurs on periplasmic side. The DEER measurements in conjunction with molecular modeling based on the x-ray structure provide strong support for the alternative access model and reveal a structure for the outward-facing conformer of LacY.conformational change ͉ double electron-electron resonance ͉ lactose permease ͉ major facilitator superfamily ͉ membrane transport T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily (MFS), utilizes free energy stored in an electrochemical H ϩ gradient (⌬˜Hϩ) to drive active transport by coupling the downhill, stoichiometric translocation of H ϩ with ⌬˜Hϩ to the uphill accumulation of galactopyranosides. In the absence of ⌬˜Hϩ, LacY can also use free energy released from downhill translocation of galactosides in either direction to drive uphill translocation of H ϩ with generation of ⌬˜Hϩ, the polarity of which depends on the direction of the sugar gradient (1, 2). An x-ray structure of LacY (3) combined with a wealth of biochemical data (1, 2, 4, 5) has led to an alternating access model for sugar translocation across the membrane. By this means, the inward-facing cytoplasmic cavity closes with opening of an outward-facing cleft, thereby making the sugar-binding site alternatively accessible to either face of the membrane. A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (6). The alternating access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (2, 7-9).A...

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“…The distance range of this approach can be extended up to at least 50 Å by applying pulse EPR techniques such as pulse electron-electron double resonance experiments (17,18) or double-quantum EPR (19). In particular, the four-pulse DEER experiment has been shown to be applicable to membrane proteins even if distances are rather broadly distributed as is the case for spin labels that are situated in loops (20). In recent methodological studies on extraction of distance distributions from four-pulse DEER data (21) and sensitivity enhancement (22), we have used doubly spin-labeled samples of LHCIIb as model systems.…”

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Localization of the N-terminal Domain in Light-harvesting Chlorophyll a/b Protein by EPR Measurements

Jeschke

Bender

Schweikardt

et al. 2005

Journal of Biological Chemistry

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The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the Nterminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.The major light-harvesting chlorophyll a/b complex (LHCIIb) 1 in higher plants contributes to photosynthesis most significantly by increasing the amount of light energy absorbed and thus of the energy flow through the photosynthetic reaction centers. This is accomplished by the pigments present in the complex, eight chlorophyll (Chl) a and six Chl b molecules as well as four carotenoids per apoprotein, which are noncovalently bound to the protein and positioned such that the excitation energy can rapidly be conducted toward the reaction centers. With regard to this function, it seems reasonable to view the LHCIIb apoprotein as a rigid scaffold keeping the pigments in their proper position.In addition to its light-harvesting functions, LHCIIb is involved in a number of regulatory processes that may require a more flexible behavior of the apoprotein. One of these processes ensures a balanced energy flow through the two photosystems (PS). When PSII reduces more plastoquinone than can be reoxidized by PSI, then LHCIIb becomes phosphorylated at a threonine residue close to the N terminus by a thylakoid kinase. This modification prompts LHCIIb to dissociate from PSII and bind to PSI, increasing the absorption cross-section of the latter (1, 2). The structural basis for this switch in binding partners is unclear, but an altered arrangement of the Nterminal hydrophilic domain in LHCIIb, triggered by its phosphorylation, has been discussed (3). Phosphory...

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Interresidual Distance Determination by Four-Pulse Double Electron-Electron Resonance in an Integral Membrane Protein: The Na+/Proline Transporter PutP of Escherichia coli

Cited by 78 publications

References 34 publications

DEER Distance Measurements on Proteins

DEER Distance Measurements on Proteins

Sugar binding induces an outward facing conformation of LacY

Localization of the N-terminal Domain in Light-harvesting Chlorophyll a/b Protein by EPR Measurements

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