nature structural & molecular biology  VOLUME 20 NUMBER 11 NOVEMBER 2013 1265

a r t i c l e s

β-barrel membrane proteins are essential components of the outer membranes of Gram-negative bacteria, mitochondria and chloro-plasts. The biogenesis of these OMPs poses a complex biophysical challenge to the cell because the OMPs are synthesized at locations distant from their target membrane1. The OMP polypeptide chains are prone to aggregation in aqueous cellular compartments and thus cannot reach their cellular destination by self-diffusion but need to be conveyed by molecular chaperones2–5. In the gram-negative bacterium E. coli, these chaperones comprise the cytosolic trigger factor and SecB, as well as the periplasmic SurA and Skp. Eventually, the OMP substrates are transported to the Bam complex, which folds and inserts them into the outer membrane6–12. Importantly, the periplasmic transport chaperones cannot rely on cellular energy and are therefore unable to use energy-dependent conformational changes for their substrate binding and release mechanisms. In con-trast, ATP-dependent chaperones, such as the chaperonins GroEL and Hsp90, modulate their affinity for substrates by energy-dependent conformational changes13,14. Chaperonins bind the substrate tightly in a high-affinity state and release it rapidly by switching to a low-affinity state15–19. The conformational states of substrates bound to GroEL and Hsp90 are typically molten globule–like states, as deter-mined by biophysical methods including NMR spectroscopy20,21. It is so far not understood at the atomic level in which state the substrate is held by a periplasmic chaperone and which molecular mechanisms facilitate substrate binding and release.

To address these questions, we determined conformations and backbone dynamics of the periplasmic chaperone Skp and its bound substrates at the atomic level. E. coli Skp is a homotrimer of 17-kDa subunits. The protein has previously been crystallized in its apo form, showing a structure of three long arms protruding from a basal

trimerization interface and forming a central cavity22–24. The biological substrate range of Skp is broad, comprising more than 30 different proteins25. Skp binds typical outer-membrane protein substrates with dissociation constants in the low-nanomolar range26. Thereby, Skp has domain-selective activity: for a bidomain substrate, the transmem-brane domain is bound by the chaperone, whereas the soluble domain protrudes from this complex and adopts its native fold24. Despite numerous studies by different biophysical methods, the conforma-tional state of the substrate could so far not be described at the atomic level27,28. For our characterizations of Skp–OMP complexes, we selected two different natural substrates, the eight-stranded β-barrels of OmpX and the transmembrane domain of (tOmpA)25,29. We deter-mined the conformational states of the two substrates bound to the chaperone and located their spatial position relative to the chaperone. These data, together with measurements of the local and the global interaction lifetimes of the OMP–chaperone complex, provide a com-prehensive description of the dynamic Skp–substrate complexes, with functional consequences for the substrate-release mechanism.

RESULTSConformation and dynamics of the Skp chaperoneUsing an improved biochemical preparation protocol that includes the denaturation and functional refolding of the chaperone during purification, we were able to produce concentrated Skp samples suit-able for high-resolution NMR experiments. Spectra of the trimeric Skp in its apo form feature a single and coherent set of resonance lines for the backbone amide moieties (Fig. 1a). In both published crystal structures of Skp (PDB 1SG2 (ref. 22) and 1U2M23), the trimeric mol-ecule assumes an asymmetric conformation; i.e., the three protomers adopt different structural conformers. The observation of a single set

Biozentrum, University of Basel, Basel, Switzerland. Correspondence should be addressed to S.H. (sebastian.hiller@unibas.ch).

Received 29 May; accepted 15 August; published online 29 September 2013; doi:10.1038/nsmb.2677

Conformation and dynamics of the periplasmic membrane-protein–chaperone complexes OmpX–Skp and tOmpA–SkpBjörn M Burmann, Congwei Wang & Sebastian Hiller

The biogenesis of integral outer-membrane proteins (OMPs) in Gram-negative bacteria requires molecular chaperones that prevent the aggregation of OMP polypeptides in the aqueous periplasmic space. How these energy-independent chaperones interact with their substrates is not well understood. We have used high-resolution NMR spectroscopy to examine the conformation and dynamics of the Escherichia coli periplasmic chaperone Skp and two of its complexes with OMPs. The Skp trimer constitutes a flexible architectural scaffold that becomes more rigid upon substrate binding. The OMP substrates populate a dynamic conformational ensemble with structural interconversion rates on the submillisecond timescale. The global lifetime  of the chaperone–substrate complex is seven orders of magnitude longer, emerging from the short local lifetimes by avidity.  The dynamic state allows for energy-independent substrate release and provides a general paradigm for the conformation of  OMP polypeptides bound to energy-independent chaperones.

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1266  VOLUME 20 NUMBER 11 NOVEMBER 2013 nature structural & molecular biology

a r t i c l e s

of resonance lines in the NMR spectra indicates that each of the three Skp protomers is sampling the same conformational space on the fast-exchange NMR time scale, i.e., within 1 ms (refs. 30,31) (Fig. 1a and Supplementary Figs. 1 and 2). Any temporary local asymmetry is averaged out on this timescale.

In a next step, we prepared stable chaperone–substrate assemblies by loading Skp with the natural substrate protein OmpX (148 aa (refs. 32,33)). Control experiments showed that the resulting complex is folding competent, and thus it is a biologically relevant on-pathway state34. For the Skp–OMP complex, the use of selective isotope-labeling schemes permitted the observation of either the chaperone or the

substrate as part of the complex by NMR spectroscopy. Spectra of holo-Skp with bound OmpX feature a single set of NMR resonance lines indicating that each of the three protomers samples the same conformational space within at most 1 ms, just as with the apo form. Importantly, binding of the large asymmetric substrate polypeptide chain does not break the trimeric conformational symmetry of Skp averaged on this timescale.

We used the chemical shifts of the backbone 13Cα and 13Cβ nuclei to identify the secondary-structure elements in apo- and holo-Skp. Apo-Skp features four α-helices and three β-strands in aqueous solution, and their number and positioning are in agreement with

a SkpS127Skp–OmpX

Skp–tOmpAI6

N120

V140

V117

V129M54 N125

K130N8

A123

A119

9.00 9.00

9.00

7.40

8.80

8.808.50 8.00 7.50 7.00

108

114

112

116

120

124

128

132

8.60

7.10 6.80

116

121

124

127

129

�2(1H) (p.p.m.)

�1(15N)(p.p.m.)

b

Skp

Skp–OmpX

Skp versus Skp–OmpX

20

∆�(13Cα)–∆�(13Cβ)(p.p.m.)

∆(∆�(13Cα)–∆�(13Cβ))(p.p.m.)

2.0

0

–2.0

2.0

0

–2.0

1.0

0

–1.0

β1 β2 β3α1 α4α2.A α2.B α3.A α3.B

40 60Skp residue number

80 100 120 140

c

J(0.87 ωH)

0–2.5 ps

apo-Skp Skp–OmpX

2.5–4.0 ps4.0–5.5 ps5.5–7.0 ps>7.0 ps

Helix α3.A

d

e

f

Skp versus Skp–OmpX

Skp residue number

Skp–tOmpA versus Skp–OmpX

∆(∆�(13Cα)–∆�(13Cβ)) (p.p.m.)

∆�(HN) (p.p.m.)

∆�(HN)(p.p.m.)

F30

N N N

CC

β3

β1

β2

α3.B

α4

α1

α2.A

α2.B

α3.AHelix α2.B

Helix α3.B

Helix α2.Aflexible

Stabilizedupon Ompbinding

0.3

0.1

0.3

0.1

20 40 60 80 100 120 140

Figure 1 Dynamic adaptation of Skp to OMP substrates. (a) Overlay of 2D 15N-1H TROSY NMR fingerprint spectra of different forms of Skp. Black, apo-Skp; green, holo-Skp with OmpX; yellow, holo-Skp with tOmpA. Sequence-specific resonance assignments are indicated. (b) Secondary backbone 13C chemical shifts of apo-Skp (black) and holo-Skp in complex with OmpX (green) and their difference (orange), plotted against the amino acid residue number of Skp. Positive values indicate α-helix and negative values β-sheet secondary- structure elements. The resulting secondary-structure assignments are indicated at top. Values >4 p.p.m. or ≤4 p.p.m. (gray lines) are shown truncated. (c) Amplitude of backbone motions on the picosecond-to-nanosecond timescale, as measured by the spectral density J(0.87ωH = 3.8 ns−1), plotted color-coded on the Skp structure (PDB 1SG2 (ref. 22)). (d) Close-up of the structural pivot element in helices α2 and α3. The flexible helix α2.A (red) is linked by the kink at residue F30, which is highly conserved among species, to the more rigid helix α2.B (yellow). The helicity of segment Ser89–Arg93 (purple) of helix α3.A (orange) is stabilized upon substrate binding. (e) Combined chemical-shift differences of the amide moieties plotted against the Skp amino acid residue number. Orange, apo-Skp versus holo-Skp with bound OmpX. Red, holo Skp with OmpX versus holo Skp with tOmpA. The magnitude of two s.d. (0.15 p.p.m.) is indicated by a dashed line. (f) Amide-group chemical-shift changes larger than two s.d. occurring upon OmpX binding are marked in cyan on the Skp structure. The segment Ser89–Arg93 of helix α3.A with increased helicity upon substrate binding is indicated in purple.

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature structural & molecular biology  VOLUME 20 NUMBER 11 NOVEMBER 2013 1267

a r t i c l e s

the crystalline structures22,23 (Fig. 1b). These seven secondary-structure elements remain preserved in number and position upon binding of the substrate OmpX, thus indi-cating that the chaperone does not undergo major structural rearrangements upon substrate binding. The only substantial difference in secondary structure between the apo and the holo form is an increase of helical population for a short seg-ment of helix α3.A. The C-terminal two turns of this helix, resi-dues Ser89–Arg93, increase in secondary chemical shift by about 0.7 p.p.m. We could thus estimate that the helical population of this segment is increased from ~80% in the apo form toward a population of ~100% in the holo form.

This stabilization of the last turns of helix 3 is correlated with a general reduction of fast backbone motions of Skp upon sub-strate binding. The amplitude of backbone dynamics depend-ent on the frequency is described by the spectral density function J(ω), which was mapped site specifically from measure-ments of the NMR relaxation parameters T1(15N), T2(15N) and 15N-1H NOE35–37 (Fig. 1c and Supplementary Fig. 3). We found that the overall molecular tumbling of the Skp chaperone is not substantially affected by binding of the substrate; however, the amplitude of motions on the picosecond-to-nanosecond timescale, J(0.87ωH), decreases substantially for large parts of the protein (Fig. 1c). This increase in rigidity is particularly evident for the long helix α3 (residues 70–103), whose averaged J(0.87ωH) value decreases from 4.7 ps in the apo to 2.5 ps in the holo form. The Skp chaperone thus undergoes a change from a more dynamic to a more rigid conformation upon binding of the OmpX substrate. The backbone dynamics show further that the short helix α2.A on the exterior of the cavity features locally increased picosecond-to- nanosecond dynamics in both the holo and apo forms, with ampli-tude averages of 5.2 ps and 5.6 ps, respectively. The two turns of helix α3.A and the helix α2.A thus form a hinge element between the arms and the basal unit, thereby facilitating the dynamic adaptation of the chaperone to its substrate (Fig. 1d).

Adaptations in structure and dynamics of the Skp chaperone to its substrate are additionally reflected in chemical-shift changes of the amide moieties. These include changes caused by direct interac-tion as well as allosterically induced chemical-shift changes, and they are distributed over most of the protein, thereby indicating a tran-sient, delocalized interaction and the absence of a localized, specific binding site (Fig. 1e,f). Importantly, the amide moieties of residues Ser53–Arg60 in the loop between helices α2 and α3 at the tip of the arms show no chemical-shift differences between the apo form and

the holo form, thus indicating that this region is not directly interact-ing with the OmpX substrate.

Equivalent experiments, including complete sequence-specific resonance assignments and measurement of backbone dynamics, were also performed for Skp holding the alternative substrate tOmpA (176 aa)25,38 (Supplementary Fig. 2). Skp shows similar adaptations in conformation and dynamics to the substrate tOmpA as to OmpX (Fig. 1a,e and Supplementary Fig. 2). In particular, the combined chemical-shift differences of the Skp amide moieties between Skp–OmpX and Skp–tOmpA are markedly smaller than are those of either of these holo forms relative to apo-Skp. The observed adaptations of Skp in conformation and dynamics to its substrates therefore repre-sent a functional mechanism intrinsic to Skp.

Conformation and dynamics of the chaperone-bound substrateIn a next phase, we addressed the structural conformation of the sub-strate protein OmpX bound to Skp. 2D 15N-1H correlation spectra of OmpX feature a single, complete set of resonances with a chemical- shift dispersion of the amide proton of about 0.8 p.p.m. (Fig. 2a). This narrow dispersion is indicative of fast conformational averag-ing locally comparable to the situation in intrinsically unstructured or chemically denatured polypeptide chains. We assessed the extent of possible secondary structure from the backbone 13C chemical shifts (Fig. 2b). These shifts feature a low deviation from random-coil values, thus indicating the absence of substantially populated elements of regular secondary structure in the conformational ensemble. The observation of averaged chemical shifts directly shows that the OmpX polypeptide chain undergoes averaging over multiple backbone conformations in the fast exchange limit; i.e., all local exchange rate constants involved in this process are >1 ms−1 (refs. 30,31).

The OmpX polypeptide thus features a high local mobility in the chaperone cavity, and this is also reflected in its spectral density function, J. For OmpX bound to Skp, the spectral density func-tion indicates essentially uniform-amplitude backbone dynamics over the entire polypeptide chain (Fig. 2c). The backbone dynam-ics on the picosecond-to-nanosecond timescale, J(0.87ωH), have a particularly large amplitude of 20 ps, which is >5 times larger than for the well-folded chaperone and is comparable to flexibly unfolded polypeptides. The substrate polypeptide thus undergoes

Figure 2 Conformation and dynamics of OmpX bound to Skp. (a) 2D 15N-1H TROSY fingerprint spectrum of [U-2H, 15N]OmpX bound to [U-2H]Skp (U denotes uniform labeling), recorded at 37 °C in NMR buffer, pH 6.5. The sequence-specific resonance assignments obtained from 3D triple resonance experiments are indicated. (b) Secondary backbone 13C chemical shifts of OmpX bound to Skp, relative to random-coil values. (c) Amplitudes of backbone motions, as measured by the spectral density function J for OmpX bound to Skp, plotted against the amino acid residue number of OmpX. Values for J are given at the frequencies 0, ωN = 500 µs−1 and 0.87ωH = 4.3 ns−1. Dashed lines indicate the sequence-averaged value at each frequency.

G8G23

G143G112

G16G22

G106

G138G55

G86 G7 G41

T102T93

T51T4 T46 S126

T139

S79

S3S130

S49S54

I44S53

S12

S35S103 S134E32

N120

N34F24

Y105D75L37

D56V135K20N60

N19M21

M18R72

K99H100R131

N116K27E128D13

A70E47

K48

L26Y30

Y127

E94

A122

A10

A52 A1

A14

A67

A142

F148

A77D136 I40 A111

R147R133

D104Y109I73Y45F43

L123

L113Y63 K89 I79

W76Y146

V121

Y71

V144

V83I132E119

Y9Y57

Y87T6

R29S108

T66

Q15F107

Q61Q17Q11

E31 M118I65V39

V85V82

W140V5

R50

G84

G145

G110

G88G38

G68

G81

G64

108

110

112

114

116

118

120

122

124

126

8.25 8.00

�2(1H) (p.p.m.)

7.75 7.50

Skp–OmpXa

�1(15H)(p.p.m.)

b∆�(13Cα)–∆�(13Cβ)(p.p.m.)

2.0

–2.0

20 40 60 80OmpX residue number

100 120 140

0

cJ(0)(ns)

16.0

12.0

4.0

8.0

J(�N)(ns)

0.3

0.2

0.1

J(0.87�H)(ps)

OmpX residue number

40

10

20 40 60 80 100 120 140

20

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1268  VOLUME 20 NUMBER 11 NOVEMBER 2013 nature structural & molecular biology

a r t i c l e s

spatial movement relative to the chaperone. At the same time, the value of J(0) is increased compared to that of an unfolded peptide, a result indicating coupling of the overall molecular tumbling to the chaperone39. Overall, the substrate adopts a conformational ensemble with rapid, submillisecond interconversion between individual conformers. Importantly, this conclusion can be safely deduced only from a sequence-specific resonance assign-ment that covers the entire polypeptide chain, as was obtained in this work.

We performed equivalent measurements of conformation and dynamics with the alternative substrate tOmpA bound to Skp (Supplementary Fig. 3). Thereby, we found the backbone dynamics, in particular the local flexibility and the absence of regular secondary-structure elements to be equivalent to that of OmpX (Supplementary Fig. 3). This observation readily suggests a gen-eral occurrence of this highly dynamic state as a conformation of Skp substrates.

Temperature-dependent line-broadening experiments gave further insights into the magnitude of the kinetic interconversion rate con-stants. At the experimental temperature of 37 °C, the rate constants for all amino acid residues in the two substrate proteins are in the fast exchange limit, i.e., above ~1 ms−1. At lower temperatures, how-ever, we observed sequence-dependent line broadening for certain polypeptide segments in each substrate protein with a concomitant signal amplitude decrease below the experimental detection limit (Fig. 3 and Supplementary Fig. 4). For these segments, the local interconversion rate constants move into the intermediate exchange regime at the lower temperature, and we thus estimate them to be just about an order of magnitude larger than 1 ms−1 at 37 °C. Notably, the location of the line-broadened segments in the polypeptides is correlated to the local hydrophilicity: The segments remaining in fast exchange at low temperature, residues T30–F40, M100–S120 and Y141–A176 in tOmpA and S3–G23 and S48–G55 in OmpX, are among the most hydrophilic polypeptide segments (Fig. 3b and Supplementary Fig. 4). Both substrate proteins thus feature a nonran-dom, local modulation of the ensemble interconversion rate constants, with hydrophobic segments having slower exchange kinetics than do hydrophilic segments.

Polypeptide chains in strong chemical denaturant solution can adopt a random-coil conformation40. We wondered how the spatial extension of the Skp-bound state compares to such an unconstrained ensemble and assessed the ensemble compactions by measurements of intramolecular paramagnetic relaxation enhancement effects (PREs). We covalently attached the paramagnetic spin label (1-oxyl-2, 2,5,5-tetramethyl-∆3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) to position 140 of the OMP polypeptide chain through a

disulfide bond. The PRE arises from dipole interactions between the unpaired electron and individual nuclear spins, thus reducing the intensity of the NMR resonances in accordance with the spin-spin distance. Owing to the strength of this interaction, the PRE is par-ticularly suited to assess spin-spin distances in the range of 15–25 Å (refs. 41–43). OmpX in 8 M urea solution is known to adopt a random-coil conformation without long-range contacts33,44 and served as a suitable reference state for a flexibly extended random-coil conformation. This form had no detectable intramolecular long-range PRE effects between position 140 and residues >20 residues away in sequence (Fig. 4a), results well in agreement with model calculations for random coils45–47. In the Skp-bound state, however, substantially large intramolecular PREs were present between position 140 and the rest of the polypeptide chain (Fig. 4a). Thereby, the average PRE of 0.26 indicates a substantially compacted conformational ensemble with extensive long-range interactions. Notably, intramolecular long-range interactions by PREs have also been observed for unrestricted polypeptides that are not in a random-coil state, such as mildly dena-tured or intrinsically unfolded proteins46,48,49. The observed PRE value for Skp-bound OmpX corresponds in first-order approximation to a polypeptide ensemble constrained to a spherical volume with a radius R of 21 Å (Fig. 4b). This ensemble contains ~50% polypeptide chain and 50% internal water molecules in the spherical volume and is thus substantially less densely packed than the smallest theoretically possible value of R of 17 Å.

A structural model of the OMP–Skp complexIn a final step, we wanted to combine our conformational descrip-tions of the well-folded chaperone and the highly dynamic substrate ensemble to a structural model of the dynamic OmpX–Skp complex. We determined their relative orientations by measurement of inter-molecular PREs between chaperone and substrate. In a first stage, we used the OmpX derivative with a single spin label at residue 140 and quantified the effect on the Skp chaperone. This paramagnetic spin label caused intermolecular PREs on Skp for most of the resi-dues lining the cavity. These are the central parts of helices α2 and

Figure 3 Differential temperature dependence of the backbone dynamics of Skp-bound tOmpA. (a) 2D 15N-1H TROSY spectra of [U-2H, 15N]tOmpA bound to [U-2H]Skp at the temperatures of 37 °C, 25 °C and 13 °C in NMR buffer at pH 6.5. Examples for amide resonances that do and do not feature line broadening in this temperature range are indicated in orange and green, respectively. (b) Local hydrophobicity of tOmpA. ∆F values are the free energies of transfer of the individual amino acids from an aqueous solution to its surface57. Hydrophobicity corresponds to negative ∆F values. Red line indicates the average value plus 0.8 s.d., the chosen threshold for identification of the most hydrophilic segments. (c) Amino acid sequence of tOmpA. The positions of the eight β-strands formed in natively folded tOmpA are indicated. Green and orange bars indicate the observable and unobservable resonances at 13 °C, respectively. Blue bars indicate the segments exhibiting the highest degree of hydrophilicity as identified in b.

β1 β2

β5β4

β7 β8

β3

β6

20

80 100

60

140

0.4

0

–0.4

∆F(kcal/mol)

20 40 60 80tOmpA residue number

100 120 140 160

�1(15N)(p.p.m.)

�2(1H) (p.p.m.)

110

37 °C

G160

T117

S163

H32 A124

W16

25 °C

G160

T117

S163

H32 A124

W16

13 °CG160

T117

S163

H32A124

W16

116

122

128

110

116

122

128

110

116

122

128

8.4 8.0 7.6 8.4 8.0 7.6 8.4 8.0 7.6

a

b

c

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature structural & molecular biology  VOLUME 20 NUMBER 11 NOVEMBER 2013 1269

a r t i c l e s

α3 and the loop between the sheets β2 and β3 (Fig. 5a). Notably, the PRE effect on the tips of helices α2 and α3 at the bottom of the chaperone and the linker between them is comparably small, thus confirming our previous observations that the tips of the chaperone arms are not in close spatial contact with the substrate. From these measurements, we obtained the average position of the spin label and thus the location of the center of the conformational substrate ensemble by geometric averaging (Fig. 5a). Our first-order model thus comprises a dynamic ensemble of conformers placed at that position (Fig. 5b).

We cross-validated the resulting structural model by intermo-lecular PREs from a paramagnetic spin on Skp on the amides of the OmpX substrate. We created three single–amino acid variants of Skp with spin labels attached at positions T60, S88 and S105 (Fig. 5b) and measured the intermolecular PRE effect on the OmpX resonances for each of them. In each case, the PRE amplitudes are distributed uniformly over the amino acid sequence of the unfolded substrate polypeptide (Fig. 5c). This averaged behavior confirms

the mobility of the substrate relative to Skp and the rapid dynamic averaging over multiple conformations. At the same time, the observed PRE amplitudes validate the structural model quantita-tively. For the spin label attached to position S105 on Skp, which cannot closely approach the OmpX ensemble, the observed average intermolecular PRE is 0.77. For the two positions S88 and T60 at the center and the tips of the arms, the hydrophobic MTSL moiety can closely interact with the OmpX ensemble, thus resulting in the value 0.25, which is of similar value to the intramolecular PRE within OmpX.

To complete the biophysical description, we determined the glo-bal lifetime of the OmpX–Skp complex. Use of a combined sample with 50% apo-Skp and 50% holo-Skp, in which just one of the species is isotope labeled, allowed observation of the equilibra-tion process between apo and holo forms by NMR spectroscopy. Exponential fitting, assuming off-rate–limited first-order kinetics, yielded an average lifetime for the OmpX–Skp complex of 2.6 ± 0.9 h (Supplementary Fig. 5).

Figure 4 Compactness of the dynamic OmpX polypeptide ensemble. (a) Intramolecular paramagnetic relaxation effect (PRE) of a paramagnetic spin label attached to [U-2H, 15N]OmpX at position W140C, plotted against the amino acid residue number of OmpX. Top, data for OmpX denatured in 8 M urea. Bottom, data for OmpX bound to Skp. The PRE is expressed as Vox/Vred, the ratio of the cross-peak volumina in 2D 15N-1H TROSY spectra before (ox) and after the chemical reduction (red) of the spin label. The dashed lines show model calculations for a flexible polypeptide in a random-coil conformation (top) and in a spherically compacted state (bottom). (b) Illustrations of the spatial compactness of the OmpX polypeptide ensembles. The random-coil conformation in 8M urea (top) and the compacted ensemble within Skp (bottom) as derived from the PRE data in a. Spheres with diameters of 46 Å and 21 Å are shown for the two cases, respectively, and a single conformer from the ensemble is shown as an example.

a1.0

b

0.6PRE(Vox/Vred) 0.2

0.6

0.2

20 40 60 80 100 120 140

21 Å

46 Å

20 40 60 80

OmpX–Skp

OmpX–Skp

OmpX in8 M urea

OmpX in8 M urea

OmpX residue number100 120 140

S105

T60

90°

b

S88

c0.8

0.4

0.8PRE(Vox/Vred) 0.4

0.8

0.4

20 60OmpX residue number

100 140

90°

a

PRE

1.0–0.80.8–0.6

0.4–0.20.6–0.4

Unassigned

Figure 5 Structural model of the Skp–OMP complex. (a) Strength of the intermolecular PRE from a paramagnetic spin label attached to OmpX W140C and detected on the amide moieties of Skp. The PRE intensities are plotted on the Skp structure (PDB 1SG2 (ref. 22)) in the indicated color code. The black sphere denotes the location of the geometric center of the affected amide moieties. A side and a bottom view are shown. (b) Structural model of the Skp–OmpX complex. A spherical volume with a radius of 21 Å (Fig. 3b, purple) is positioned at the geometric center determined in a. The spherical volume indicates the spatial position of the bound OmpX ensemble in first-order approximation. The positions for the spin labels used to cross-validate this model are shown in ball-and-stick representation. (c) Strength of the intermolecular PRE effect, plotted as the peak volume ratio Vox/Vred versus the amino acid residue number of OmpX. Data are shown for three different spin labels attached to Skp: blue, Skp S105C; silver, Skp S88C; brown, Skp T60C.

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1270  VOLUME 20 NUMBER 11 NOVEMBER 2013 nature structural & molecular biology

a r t i c l e s

DISCUSSIONOmpX–Skp is a biologically functional protein–protein complex con-sisting of a well-folded protein and a highly dynamic, unfolded pro-tein. Its structural description is given in first order by an ensemble of individual conformers inside the Skp cavity that stand in chemical equilibrium (Fig. 6a). In the conformational ensemble, the lifetime of any substrate conformation is <1 ms, as evidenced by the observation of fast chemical-shift averaging. However, with ~2.6 h, the global life-time of the Skp–OMP complex is seven orders of magnitude longer. An OMP molecule thus samples >107 different conformations before it leaves the Skp cavity. The magnitude of difference of these life-times reveals the avidity effect as the underlying mechanism for the high-affinity interaction between chaperone and substrate. At any given time, the substrate forms a multitude of weak local interac-tions of individual segments of the substrate polypeptide with the chaperone. Each of these interactions is transient, with a lifetime of 1 ms or less. These weak affinities add up to the high global binding affinity with a dissociation constant in the low-nanomolar range, as observed for typical Skp substrates26. Importantly, these weak, transient interactions of the individual OMP polypeptide segments are sequence unspecific, thus allowing Skp to interact in a general fashion with a broad range of different substrates and also leaving room for evolutionary changes in the amino acid sequence of the substrates25,50.

The fast backbone dynamics of the Skp–substrate complex also provides a rationale for the thermodynamics of the OMP polypeptide transport toward downstream folding. The bound OMP substrates are held by Skp in a flexible ensemble and thus a thermodynamic equilib-rium state high in conformational entropy and low in enthalpy. These properties enable the subsequent exergonic folding of the substrate protein into the outer membrane, a process driven by the enthalpy gains of barrel folding and membrane insertion, in excellent agree-ment with current thermodynamic investigations51,52.

The flexibility of the Skp-bound OMP substrate also facilitates its release to a downstream receptor. The weak local affinities and the rapid structural interconversion can well allow simultaneous inter-actions of segments of the substrate polypeptide chain with other proteins while an OMP is still partially bound to Skp. For example, if a downstream receptor has a specific affinity for a certain polypeptide segment that is higher than the unspecific affinity of the same segment

for Skp, the downstream receptor could readily select this segment out of the conformational ensemble. Intriguingly, this scenario is met by the ‘β-signal’, a functionally conserved polypeptide segment in all integral OMPs, recognized by the membrane-inserting Bam com-plex53–56. It is thus tempting to speculate that a transition complex could be formed (as shown schematically in Fig. 6b) in which the dynamic Skp–OMP complex would present the β-signal transiently to Bam, which would then catalyze the subsequent folding and mem-brane insertion of the OMP. In contrast, if the OMP-chaperone inter-action would be nondynamic and nondelocalized, it would lead to a trapped complex and, in the absence of cellular energy, to a dead end in OMP biogenesis. For the transition to a downstream receptor, the avidity-based interaction would also allow for fast release kinetics of the substrate in the millisecond timescale without the need for a conformational change of Skp because the release rate of the flexible substrate is limited by the local and not the global lifetime. Overall, the dynamic properties of the OMP–Skp complex revealed here are thus well consistent with the biological and biophysical requirements for Skp function.

The dynamic ensemble of the two unfolded OMP polypeptide chains bound to Skp is a unique conformational state for protein polypeptides. The Skp-bound states feature submillisecond backbone dynamics, a spatial compaction and no regular secondary-structure elements. These properties, in particular the fast backbone dynam-ics and the absence of secondary structure, differ substantially from molten-globule or molten globule–like states that have been found for substrates bound to the ATP-dependent chaperones GroEL and Hsp90 (refs. 20,21). At the same time, the Skp-bound substrate has properties that differ also from the confined unfolded state that we used in first-order approximation to describe its conformation. The sequence-dependent local variations of the backbone dynamics at lower temperatures are a deviation from random behavior and cannot be expected to occur in a spherically constrained ensemble of random conformers. For the description of the unique Skp-bound state of the OMP polypeptides, and in particular to underline the contrast to the molten-globule or molten globule–like states found for substrates in other chaperones, we introduce the term ‘fluid globule’.

It is well possible that other energy-independent transport chaper-ones hold their substrates in a similarly fluidic conformational state ensemble. To assess this possibility, we have recorded the fingerprint spectra of tOmpA bound to three other ATP-independent chaper-ones from E. coli, the periplasmic SurA and the cytosolic SecB and trigger factor. For each of them, we obtained 2D NMR spec-tra with averaged chemical shifts and partially similar peak pat-terns as for Skp (Supplementary Fig. 6). Even though these other membrane protein–chaperone systems will require more detailed studies, these data are consistent with the occurrence of fluid globule– like states.

a �complex = 2.6 h

�1 < 1 ms

Outer membrane Bam

β-signal

β-signal

�3 < 1 ms�2 < 1 ms

bFigure 6 Ensemble equilibrium and biological context of the Skp–OMP complex. (a) Graphical representation of the conformational equilibrium of Skp–OmpX and Skp–tOmpA complexes. The complex is an ensemble of interconverting conformations in fast equilibrium with a global lifetime τcomplex of 2.6 h. Each individual conformation i has a lifetime τi of <1 ms. For this illustration, randomly generated OmpX polypeptide conformations (purple) were positioned into the Skp structure in ribbon representation (PDB 1SG2, green), according to the constraints determined in Figures 3 and 4. (b) Schematic model of a hypothetical transition complex of OMP–Skp (purple and green, respectively) with the downstream receptor Bam (gray). The β-signal within the OMP polypeptide chain, which is part of all natural Bam substrates, is indicated.

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature structural & molecular biology  VOLUME 20 NUMBER 11 NOVEMBER 2013 1271

a r t i c l e s

METHODSMethods and any associated references are available in the online version of the paper.

Accession codes. Sequence-specific resonance assignments have been submitted to the Biological Magnetic Resonance Data Bank under the following accession codes: apo-Skp, 19407; holo-Skp with bound OmpX, 19408; holo-Skp with bound tOmpA, 19409; OmpX bound to Skp, 19411; and tOmpA bound to Skp, 19410.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACknoWledgMentSWe thank R. Horst, P. Schanda, A. Gossert and G. Wider for discussions and D. Kahne (Harvard University) for the SurA plasmid. This work was supported by grants from the Swiss National Science Foundation (grant PP00P3_128419) and from the European Research Commission (FP7 contract MOMP 281764) to S.H. and by personal fellowships from the Novartis Foundation to B.M.B. and from the Werner-Siemens Foundation to C.W.

AUtHoR ContRIBUtIonSB.M.B. and S.H. designed the study, analyzed the data, discussed the results and wrote the paper. B.M.B. and C.W. conducted the paramagnetic spin label experiments, and B.M.B. conducted all other experimental work.

CoMPetIng FInAnCIAl InteReStSThe authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Rigel, N.W. & Silhavy, T.J. Making a β-barrel: assembly of outer membrane proteins in Gram-negative bacteria. Curr. Opin. Microbiol. 15, 189–193 (2012).

2. Ruiz, N., Kahne, D. & Silhavy, T.J. Advances in understanding bacterial outer-membrane biogenesis. Nat. Rev. Microbiol. 4, 57–66 (2006).

3. Neupert, W. & Herrmann, J.M. Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 (2007).

4. Knowles, T.J., Scott-Tucker, A., Overduin, M. & Henderson, I.R. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nat. Rev. Microbiol. 7, 206–214 (2009).

5. Schleiff, E. & Becker, T. Common ground for protein translocation: access control for mitochondria and chloroplasts. Nat. Rev. Mol. Cell Biol. 12, 48–59 (2011).

6. Hagan, C.L., Silhavy, T.J. & Kahne, D. β-Barrel membrane protein assembly by the Bam complex. Annu. Rev. Biochem. 80, 189–210 (2011).

7. Oh, E. et al. Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147, 1295–1308 (2011).

8. Bechtluft, P., Nouwen, N., Tans, S.J. & Driessen, A.J. SecB: a chaperone dedicated to protein translocation. Mol. Biosyst. 6, 620–627 (2010).

9. Zimmer, J., Nam, Y.S. & Rapoport, T.A. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature 455, 936–943 (2008).

10. Driessen, A.J. & Nouwen, N. Protein translocation across the bacterial cytoplasmic membrane. Annu. Rev. Biochem. 77, 643–667 (2008).

11. Merdanovic, M., Clausen, T., Kaiser, M., Huber, R. & Ehrmann, M. Protein quality control in the bacterial periplasm. Annu. Rev. Microbiol. 65, 149–168 (2011).

12. Voulhoux, R., Bos, M.P., Geurtsen, J., Mols, M. & Tommassen, J. Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299, 262–265 (2003).

13. Horwich, A.L. & Fenton, W.A. Chaperonin-mediated protein folding: using a central cavity to kinetically assist polypeptide chain folding. Q. Rev. Biophys. 42, 83–116 (2009).

14. Jackson, S.E. Hsp90: structure and function. Top. Curr. Chem. 328, 155–240 (2013).

15. Fang, Y., Fliss, A.E., Robins, D.M. & Caplan, A.J. Hsp90 regulates androgen receptor hormone binding affinity in vivo. J. Biol. Chem. 271, 28697–28702 (1996).

16. Roseman, A.M., Chen, S., White, H., Braig, K. & Saibil, H.R. The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell 87, 241–251 (1996).

17. Clare, D.K. et al. ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin. Cell 149, 113–123 (2012).

18. Hessling, M., Richter, K. & Buchner, J. Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90. Nat. Struct. Mol. Biol. 16, 287–293 (2009).

19. Street, T.O., Lavery, L.A. & Agard, D.A. Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone. Mol. Cell 42, 96–105 (2011).

20. Horst, R. et al. Direct NMR observation of a substrate protein bound to the chaperonin GroEL. Proc. Natl. Acad. Sci. USA 102, 12748–12753 (2005).

21. Park, S.J., Borin, B.N., Martinez-Yamout, M.A. & Dyson, H.J. The client protein p53 adopts a molten globule–like state in the presence of Hsp90. Nat. Struct. Mol. Biol. 18, 537–541 (2011).

22. Korndörfer, I.P., Dommel, M.K. & Skerra, A. Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture. Nat. Struct. Mol. Biol. 11, 1015–1020 (2004).

23. Walton, T.A. & Sousa, M.C. Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation. Mol. Cell 15, 367–374 (2004).

24. Walton, T.A., Sandoval, C.M., Fowler, C.A., Pardi, A. & Sousa, M.C. The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains. Proc. Natl. Acad. Sci. USA 106, 1772–1777 (2009).

25. Jarchow, S., Luck, C., Gorg, A. & Skerra, A. Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone Skp. Proteomics 8, 4987–4994 (2008).

26. Qu, J., Mayer, C., Behrens, S., Holst, O. & Kleinschmidt, J.H. The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions. J. Mol. Biol. 374, 91–105 (2007).

27. Hong, H. & Tamm, L.K. Elastic coupling of integral membrane protein stability to lipid bilayer forces. Proc. Natl. Acad. Sci. USA 101, 4065–4070 (2004).

28. Bulieris, P.V., Behrens, S., Holst, O. & Kleinschmidt, J.H. Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide. J. Biol. Chem. 278, 9092–9099 (2003).

29. Denoncin, K., Schwalm, J., Vertommen, D., Silhavy, T.J. & Collet, J.F. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics. Proteomics 12, 1391–1401 (2012).

30. Wüthrich, K. NMR assignments as a basis for structural characterization of denatured states of globular proteins. Curr. Opin. Struct. Biol. 4, 93–99 (1994).

31. McConnell, H.M. Reaction rates by nuclear magnetic resonance. J. Chem. Phys. 28, 430–431 (1958).

32. Vogt, J. & Schulz, G.E. The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence. Structure 7, 1301–1309 (1999).

33. Hiller, S., Wider, G., Imbach, L.L. & Wüthrich, K. Interactions with hydrophobic clusters in the urea-unfolded membrane protein OmpX. Angew. Chem. Int. Ed. Engl. 47, 977–981 (2008).

34. Burmann, B.M. & Hiller, S. Solution NMR studies of membrane-protein-chaperone complexes. Chimia 66, 759–763 (2012).

35. Bracken, C., Carr, P.A., Cavanagh, J. & Palmer, A.G. III. Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA. J. Mol. Biol. 285, 2133–2146 (1999).

36. Dyson, H.J. & Wright, P.E. Nuclear magnetic resonance methods for elucidation of structure and dynamics in disordered states. Methods Enzymol. 339, 258–270 (2001).

37. Peng, J.W. & Wagner, G. Mapping of spectral density-functions using heteronuclear NMR relaxation measurements. J. Magn. Reson. 98, 308–332 (1992).

38. Pautsch, A. & Schulz, G.E. Structure of the outer membrane protein A transmembrane domain. Nat. Struct. Biol. 5, 1013–1017 (1998).

39. Viles, J.H. et al. Local structural plasticity of the prion protein: analysis of NMR relaxation dynamics. Biochemistry 40, 2743–2753 (2001).

40. Fleming, P.J. & Rose, G.D. in Protein Folding Handbook (eds. Buchner, J. & Kiefhaber, T.) 706–732 (Wiley, 2004).

41. Solomon, I. Relaxation processes in a system of two spins. Phys. Rev. 99, 559–565 (1955).

42. Bloembergen, N. & Morgan, L.O. Proton relaxation times in paramagnetic solutions: effects of electron spin relaxation. J. Chem. Phys. 34, 842–850 (1961).

43. Battiste, J.L. & Wagner, G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. Biochemistry 39, 5355–5365 (2000).

44. Tafer, H., Hiller, S., Hilty, C., Fernández, C. & Wüthrich, K. Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX). Biochemistry 43, 860–869 (2004).

45. Flory, P.J. Statistical Mechanics of Chain Molecules (Oxford University Press, 1989).

46. Lietzow, M.A., Jamin, M., Dyson, H.J. & Wright, P.E. Mapping long-range contacts in a highly unfolded protein. J. Mol. Biol. 322, 655–662 (2002).

47. Felitsky, D.J., Lietzow, M.A., Dyson, H.J. & Wright, P.E. Modeling transient collapsed states of an unfolded protein to provide insights into early folding events. Proc. Natl. Acad. Sci. USA 105, 6278–6283 (2008).

48. Teilum, K., Kragelund, B.B. & Poulsen, F.M. Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling. J. Mol. Biol. 324, 349–357 (2002).

49. Huang, J.R. & Grzesiek, S. Ensemble calculations of unstructured proteins constrained by RDC and PRE data: a case study of urea-denatured ubiquitin. J. Am. Chem. Soc. 132, 694–705 (2010).

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1272  VOLUME 20 NUMBER 11 NOVEMBER 2013 nature structural & molecular biology

50. Chen, R. & Henning, U. A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins. Mol. Microbiol. 19, 1287–1294 (1996).

51. Wu, S. et al. Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism. Biochem. J. 438, 505–511 (2011).

52. Moon, C.P., Zaccai, N.R., Fleming, P.J., Gessmann, D. & Fleming, K.G. Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm. Proc. Natl. Acad. Sci. USA 110, 4285–4290 (2013).

53. Robert, V. et al. Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif. PLoS Biol. 4, e377 (2006).

54. Kutik, S. et al. Dissecting membrane insertion of mitochondrial β-barrel proteins. Cell 132, 1011–1024 (2008).

55. Ieva, R. & Bernstein, H.D. Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane. Proc. Natl. Acad. Sci. USA 106, 19120–19125 (2009).

56. Hagan, C.L., Kim, S. & Kahne, D. Reconstitution of outer membrane protein assembly from purified components. Science 328, 890–892 (2010).

57. Bull, H.B. & Breese, K. Surface tension of amino acid solutions: a hydrophobicity scale of the amino acid residues. Arch. Biochem. Biophys. 161, 665–670 (1974).

a r t i c l e snp

g©

201

3 N

atur

e A

mer

ica,

Inc.

All

right

s re

serv

ed.

nature structural & molecular biologydoi:10.1038/nsmb.2677

ONLINE METHODSCloning, expression and purification of Skp. Skp lacking its signal sequence was cloned from genomic DNA through NdeI and XhoI into the pET28b expres-sion vector (Novagen) containing a thrombin-cleavable N-terminal His6 tag. BL21(λ DE3)pLysS cells (Novagen) were transformed with the plasmid and grown at 37 °C in medium containing 30 µg/ml kanamycin to OD600 = 0.6 and then for an additional 30 min at 20 °C. Expression was induced by 0.4 mM IPTG. Cells were harvested 15–18 h after induction, resuspended in buffer A (25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM imidazole and Complete EDTA-free protease inhibitor (Roche)) at a 4:1 buffer/pellet weight ratio and lysed by four passes through a French press. The lysate was centrifuged for 30 min at 12,000g at 4 °C, applied to a Ni2+-HisTrap column (GE Healthcare) and eluted by an imidazole gradient. Skp eluted at 300 mM imidazole concentration. The elution fractions containing Skp were concentrated in a Vivaspin 10-kDa-cutoff con-centrator (Vivascience). Concentrated Skp was denatured with 6 M Gdm/HCl, applied to Ni2+ beads (Genscript), and eluted with 300 mM imidazole. The eluted Skp was dialyzed overnight against assembly buffer (25 mM HEPES, pH 7.5, and 150 mM NaCl). Afterwards, Skp was concentrated by ultrafiltration and stored at −20 °C until use.

Cloning, expression and purification of SecB, trigger factor and SurA. SecB and trigger factor were cloned from genomic DNA into pET28b. The SurA plas-mid obtained from Daniel Kahne (Harvard University)56 was cloned with the same strategy and lacked its signal sequence. Expression and purification was done as described for Skp.

Cloning, expression and purification of tOmpA and OmpX. The transmem-brane domain of OmpA (residues 1–177) and OmpX were cloned through NcoI and XhoI into the pET28b expression vector without any affinity tag and lack-ing their respective signal sequence. BL21(λ DE3)pLysS cells were transformed with the tOmpA or OmpX expression plasmid and grown at 37 °C in medium containing 30 µg/ml kanamycin to OD600 = 0.8. Expression was induced by 1 mM IPTG. Cells were harvested 4 h after induction, resuspended in buffer B (20 mM Tris-HCl and 5 mM EDTA, pH 8.5) at a 4:1 buffer/pellet weight ratio and lysed by four passes through a French press. Purification from inclusion bodies was done for both proteins as described for OmpX44. The ion-exchange elution fractions containing tOmpA or OmpX were pooled and dialyzed against buffer B. The precipitate was resuspended in 6 M Gdm/HCl and stored at −20 °C until usage. Final yields of purified protein were 30 mg OmpX or 50 mg tOmpA per liter of deuterated M9 minimal medium.

Isotope labeling. We obtained [U-2H, 15N]- and [U-2H, 15N, 13C]-labeled OMPs by growing the expression cells in M9 minimal media58 supplemented with (15NH4)Cl, d-[2H, 13C]glucose and D2O. [U-2H]Skp was obtained by the addition of d-[2H]glucose and D2O to M9 minimal medium. All isotopes were purchased from Sigma-Aldrich.

Mutagenesis, expression and purification of Skp and OmpX single-cysteine mutants. The QuikChange II mutagenesis protocol (Stratagene) was used to introduce the mutation T60C, S88C or S105C into Skp or W140C into OmpX. PCR primers were obtained from Microsynth. The expression and purification of the mutant proteins was performed as described for the wild-type proteins, except that 1 mM DTT was added to all buffers.

Site-specific spin labeling of cysteine mutants. Spin labeling of the cysteines introduced into Skp and OmpX, with MTSL ((1-Oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl)-methanethiosulfonate; Toronto Research Chemicals) was done according to published protocols59,60. Protein solution in assembly buffer containing DTT was exchanged to assembly buffer without DTT in Vivaspin 5-kDa concentrators (Vivascience). A ten-fold molar excess of MTSL dissolved in acetonitrile was added to the protein solution, and this was fol-lowed by incubation overnight at room temperature in the dark. To remove unreacted MTSL, the buffer was exchanged to NMR buffer (25 mM MES and 150 mM NaCl, pH 6.5). For reduction of the spin label, ascorbate was added directly to the NMR tube from a 500-mM stock solution to a final concentration of 5 mM.

Complex assembly. An excess of denatured tOmpA or OmpX was added to Skp in assembly buffer in a drop-wise fashion under continuous stirring at 4 °C. To ensure saturation of the chaperones, the OMP was added until precipitation was observable. The solution was then stirred for another 2 h. After centrifugation at 10,000g for 30 min, the supernatant was concentrated by ultrafiltration, and assembly buffer was exchanged to NMR buffer.

NMR spectroscopy. All NMR experiments for Skp–OMP complexes were per-formed in NMR buffer. Urea-denatured OmpX was measured in 20 mM sodium phosphate and 8M urea, pH 4.0. The experiments were recorded at 310 K for the Skp–OMP complexes and at 283 K for urea-denatured OmpX W140C on Bruker AscendII 700 MHz and Avance 800 MHz spectrometers with a cryo-genically cooled triple-resonance probe. For the sequence-specific backbone resonance assignment of [U-99% 2H, 13C, 15N]-OMP bound to [U-99% 2H]Skp, the following NMR experiments were recorded: 2D 15N-1H TROSY-HSQC61, 3D TROSY-HNCA, 3D TROSY-HNCACB, 3D TROSY-HNCO, and 3D TROSY-HN(CA)CO62. For the sequence-specific backbone resonance assignment of [U-99% 2H, 13C, 15N]Skp with and without bound OmpX, the same set of experiments as for the OMPs in the chaperone-bound state with the addition of 3D 1H-1H NOESY–15N TROSY experiments63–65 with a mixing time of 100 ns were recorded. For determination of the dynamic properties of [U-95% 2H, 15N]OMP bound to unlabeled Skp and for apo and holo [U-99% 2H, 13C, 15N]Skp bound to [U-99% 2H]OmpX and [U-99% 2H]tOmpA, respectively, the following experiments were measured: 15N-1H NOE66, T1 (ref. 66), T1ρ (ref. 67), TRACT15,68 and N-filtered BPP-LED-diffusion (Supplementary Table 1)69. For the determination of the lifetime of the Skp–OmpX complex, 150 µM of unla-beled Skp–OmpX were mixed with a stoichiometric amount of [U-2H, 15N]Skp at t = 0. Subsequently, a series of 2D 15N-1H TROSY fingerprint spectra with experimental length of 230 min was recorded. The volumes of a selected set of resonance peaks of the apo and holo form were integrated. Nonlinear least-squares fitting of a double exponential function yielded the global lifetime con-stant of the Skp–OmpX complex. NMR data were processed with PROSA70 and analyzed with CARA and XEASY71. Nonlinear least-square fits of relaxation data were done with Matlab (MathWorks). T2 values were derived from T1ρ (ref. 72). Error bars for T1 and T2 were calculated by a statistical bootstrapping scheme and error bars for the 15N{1H} NOE and for the PRE measurements from the spectral noise. Error bars for the spectral density function were obtained by error propagation. Secondary chemical shifts were calculated relative to the random-coil values of Kjaergaard and Poulsen73.

Numerical calculations of PREs. The paramagnetic relaxation effect (PRE) of an electron spin on a nuclear spin is given by

Γ20

22 21

20 44 0 3=

+mp

m g wB2

H Hg J J( ( ) ( ))

where µ0 is the magnetic permeability of the vacuum, µB the Bohr magneton, g the electron spin g factor, γH the proton gyromagnetic ratio and ωH the proton resonance frequency in angular units. The spectral density function is given by

Jr

( )w tw t

= 16 2

r2

r1 +

where r is the interspin distance and τr an effective rotational correlation time. The cornered brackets denote time and ensemble average. This ‘Gillespie–Shortle’ model has been shown to be applicable to unfolded proteins74,75. For model calcu-lations of the random flight chain, r is given by the expectation value for a random flight chain ⟨ ⟩r Nl2 2= , where N is the distance in residues from the spin label, and l is the length of an amino acid residue backbone, 3.8 Å (ref. 45). The value τr = 4.0 ns was obtained by fitting of the model calculations to the experimental data of urea-denatured OmpX. Restricted codiffusion in a sphere is given by

16 1 2 1 2r

x x f x x= −∫ ∫d d

( )

where the two integrals run over a spherical volume with radius R, and

f yy

( )| |

= 16

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature structural & molecular biology doi:10.1038/nsmb.2677

if | |y D> and f y( )

= 0, if | |

y D≤ , where D is the minimal interspin dis-

tance due to steric hindrance. The relation between volume ratio and PRE is given by V Vox red/ exp( ( ) )= − ⋅Γ2

1 2HNIt , where τI is the length of the

INEPT element74.

58. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, 1989).

59. Berliner, L.J., Grunwald, J., Hankovszky, H.O. & Hideg, K. A novel reversible thiol-specific spin label: papain active site labeling and inhibition. Anal. Biochem. 119, 450–455 (1982).

60. Burmann, B.M., Scheckenhofer, U., Schweimer, K. & Rösch, P. Domain interactions of the transcription-translation coupling factor Escherichia coli NusG are intermolecular and transient. Biochem. J. 435, 783–789 (2011).

61. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl. Acad. Sci. USA 94, 12366–12371 (1997).

62. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wüthrich, K. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl. Acad. Sci. USA 95, 13585–13590 (1998).

63. Xia, Y., Sze, K.H. & Zhu, G. Transverse relaxation optimized 3D and 4D 15N/15N separated NOESY experiments of 15N labeled proteins. J. Biomol. NMR 18, 261–268 (2000).

64. Zuiderweg, E.R.P. & Fesik, S.W. Heteronuclear 3-dimensional NMR-spectroscopy of the inflammatory protein C5a. Biochemistry 28, 2387–2391 (1989).

65. Marion, D. et al. Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1β. Biochemistry 28, 6150–6156 (1989).

66. Zhu, G., Xia, Y.L., Nicholson, L.K. & Sze, K.H. Protein dynamics measurements by TROSY-based NMR experiments. J. Magn. Reson. 143, 423–426 (2000).

67. Szyperski, T., Luginbühl, P., Otting, G., Güntert, P. & Wüthrich, K. Protein dynamics studied by rotating frame 15N spin relaxation-times. J. Biomol. NMR 3, 151–164 (1993).

68. Lee, D., Hilty, C., Wider, G. & Wüthrich, K. Effective rotational correlation times of proteins from NMR relaxation interference. J. Magn. Reson. 178, 72–76 (2006).

69. Chou, J.J., Baber, J.L. & Bax, A. Characterization of phospholipid mixed micelles by translational diffusion. J. Biomol. NMR 29, 299–308 (2004).

70. Güntert, P., Dötsch, V., Wider, G. & Wüthrich, K. Processing of multi-dimensional NMR data with the new software PROSA. J. Biomol. NMR 2, 619–629 (1992).

71. Bartels, C., Xia, T.H., Billeter, M., Güntert, P. & Wüthrich, K. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR 6, 1–10 (1995).

72. Walton, T.A., Sandoval, C.M., Fowler, C.A., Pardi, A. & Sousa, M.C. The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains. Proc. Natl. Acad. Sci. USA 106, 1772–1777 (2009).

73. Kjaergaard, M. & Poulsen, F.M. Sequence correction of random coil chemical shifts: correlation between neighbor correction factors and changes in the Ramachandran distribution. J. Biomol. NMR 50, 157–165 (2011).

74. Gillespie, J.R. & Shortle, D. Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. paramagnetic relaxation enhancement by nitroxide spin labels. J. Mol. Biol. 268, 158–169 (1997).

75. Xue, Y. et al. Paramagnetic relaxation enhancements in unfolded proteins: theory and application to drkN SH3 domain. Protein Sci. 18, 1401–1424 (2009).

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.