Solution NMR Structure of Proteorhodopsin

Reckel, Sina; Gottstein, Daniel; Stehle, Jochen; Löhr, Frank; Verhoefen, Mirka-Kristin; Takeda, Mitsuhiro; Silvers, Robert; Kainosho, Masatsune; Glaubitz, Clemens; Wachtveitl, Josef; Bernhard, Frank; Schwalbe, Harald; Güntert, Peter; Dötsch, Volker

doi:10.1002/ange.201105648

Cited by 31 publications

(42 citation statements)

References 42 publications

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“…PREs, generated by attachment of a spin label to a reactive cysteine in a protein, are powerful structural constraints that have been used in a large number of structural studies of both folded (22,23) and unfolded proteins (24)(25)(26)(27)(28) and to generate atomic models of molecular complexes (29,30). The r −6 dependence of the PRE on the distance between the unpaired electron of the spin label and the affected NMR probe provides long range information that extends up to distances of 25-30 Å (31), significantly larger than those obtained from nuclear Overhauser effect (NOE) measurements.…”

Section: Resultsmentioning

confidence: 99%

Hsp70 biases the folding pathways of client proteins

Sekhar

Rosenzweig

Bouvignies

et al. 2016

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

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The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a 1 H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.Hsp70 | protein folding | excited states | molecular chaperones | PRE T he 70-kDa heat shock protein (Hsp70) chaperone system is an important component of the cellular proteostasis machinery, serving as a central hub to channel client proteins along folding, refolding, maturation, disaggregation, and proteolytic pathways in cooperation with other chaperone assemblies such as Hsp90, Hsp104, and GroEL/ES (1-3). Central to Hsp70 function is its ATP-dependent interaction with client proteins, facilitated by Hsp40 cochaperones and nucleotide exchange factors (NEFs) (4). Hsp70 is a weak ATPase that recognizes and binds substrates at sites containing large aliphatic hydrophobic residues, Ile, Leu, and Val, flanked by positively charged amino acids such as Arg and Lys (5). Initial binding of substrate to the ATP-form of Hsp70 can occur directly, or via Hsp40, with rapid on/off kinetics that give rise to a weak overall affinity for the interaction. Subsequent ATP hydrolysis, stimulated by interactions with Hsp40 and substrate, leads to a large conformational change in the chaperone, locking the substrate in the Hsp70 bound state. The resulting complex is of high affinity with slow substrate on/off rates (2).Escherichia coli DnaK is the best studied of Hsp70 chaperones. It is a 70-kDa protein comprised of an N-terminal ATPase and a C-terminal substrate binding domain that communicate allosterically to couple ATP hydrolysis with substrate binding (3). Highresolution structures of ADP-(6) and ATP-DnaK (7, 8) establish that these two domains dock on to one another in the ATP-DnaK state, but become detached from each other in the ADP-bound form. In contrast to the detailed structural studies characterizing DnaK, little atomic...

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Section: Resultsmentioning

confidence: 99%

Hsp70 biases the folding pathways of client proteins

Sekhar

Rosenzweig

Bouvignies

et al. 2016

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

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“…Ideally, the location of the splicing site should have only minimal effects on protein structure or function. We therefore selected the BC loop because no distinct functional role was known so far, and only a small ␤ turn in contrast to the full ␤ sheet, as in bacteriorhodopsin, has been observed (18,19,30,31). Other regions, such as the EF loop, are known to affect color and protein stability (22,32), none of which has been reported for the BC loop.…”

Section: Resultsmentioning

confidence: 99%

“…Vector Design and Protein Expression-The N segment containing the helices A and B of proteorhodopsin without its signal sequence (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and the DnaE Npu N102 (Addgene, pSABAD219) were cloned in a pRSF vector (Invitrogen). The C segment, constructed of an N-terminal Smt3 solubility tag (UniProt, Q12306), helices C, D, E, F, and G of pR, Npu ⌬36 , and a C-terminal HSV-His 6 tag for purification, were cloned into a pBAD vector (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…Structural data of pR (18,19,31) indicate a ␤ turn motif in the BC loop. Although different studies suggest contradictory data for the exact position of this ␤ turn (Trp 83 -Gly 87 (19) and Gly 87 -Pro 90 (18)), the introduced mutations are in or next to the turn, respectively.…”

Section: Data In the Context Of Folding And Assembly Of ␣-Helicalmentioning

confidence: 99%

“…The pR protein family can be divided into green-and blue-absorbing subgroups (17). The backbone structure of green pR in detergent micelles is known from solution-state NMR (18), whereas the structure of blue pR was solved by x-ray crystallography (19). Furthermore, the functional mechanism of pR has been studied extensively by optical and infrared spectroscopy, electrophysiology, and solid-state NMR (20 -26).…”

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confidence: 99%

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Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

Mehler¹,

Eckert

Busche³

et al. 2015

Journal of Biological Chemistry

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Background: Protein trans-splicing as a molecular design tool has been demonstrated for soluble but not yet for membrane proteins. Results: Two separate polypeptides have been spliced in vivo, yielding correctly folded and functional proteorhodopsin. Conclusion: Trans-splicing of ␣-helical membrane proteins under native conditions is possible. Significance: Our findings are important for the folding, assembly, and engineering of membrane proteins.

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Die ersten Strukturen von 7‐Helix‐Transmembranproteinen in Lösung

Zerbe

2011

Angewandte Chemie

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Herausforderung gemeistert: Die Strukturaufklärung des 7‐Helix‐Transmembranproteins Proteorhodopsin in Lösung gelang kürzlich mithilfe NMR‐spektroskopischer Methoden auf der Basis von klassischen NOEs in Kombination mit paramagnetischen Relaxationverstärkungen (PREs) und dipolaren Restkopplungen (RDCs) (siehe Bild). Die Ergebnisse bedeuten einen großen Fortschritt für die Strukturbiologie.

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Solution NMR Structure of Proteorhodopsin

Cited by 31 publications

References 42 publications

Hsp70 biases the folding pathways of client proteins

Hsp70 biases the folding pathways of client proteins

Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

Die ersten Strukturen von 7‐Helix‐Transmembranproteinen in Lösung

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