Journal of Magnetic Resonance 243 (2014) 93–97
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Detection of a transient intramolecular hydrogen bond using 1JNH scalar couplings ShengQi Xiang a, Markus Zweckstetter a,b,c,⇑ a
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany c Centre for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany b
a r t i c l e
i n f o
Article history: Received 16 December 2013 Revised 4 April 2014 Available online 18 April 2014 Keywords: NMR spectroscopy Scalar coupling Hydrogen bond Folding
a b s t r a c t Hydrogen bonds are essential for the structure, stability and folding of proteins. The identiﬁcation of intramolecular hydrogen bonds, however, is challenging, in particular in transiently folded states. Here we studied the presence of intramolecular hydrogen bonds in the folding nucleus of the coiled-coil structure of the GCN4 leucine zipper. Using one-bond 1JNH spin–spin coupling constants and hydrogen/deuterium exchange, we demonstrate that a transient intramolecular hydrogen bond is present in the partially helical folding nucleus of GCN(16–31). The data demonstrate that 1JNH couplings are a sensitive tool for the detection of transient intramolecular hydrogen bonds in challenging systems where the effective/useable protein concentration is low. This includes peptides at natural abundance but also uniformly labeled biomolecules that are limited to low concentrations because of precipitation or aggregation. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Hydrogen bonds are essential for the structure and stability of many biochemical compounds. Protein folding, the formation of amyloid aggregates, enzymatic catalysis, drug–receptor interactions, and many other phenomena are intrinsically connected to hydrogen bonding [1–3]. However, identiﬁcation of hydrogen bonds in proteins is challenging, since hydrogen atoms are visible only in highest-resolution X-ray data. On the other hand, several nuclear spin properties – such as hydrogen/deuterium (H/D) exchange rates , temperature coefﬁcients , chemical shifts anisotropy , and quadrupole coupling constants  – are sensitive to hydrogen bond formation and can therefore be used to probe intramolecular hydrogen bonds using NMR spectroscopy. NMR-based measurement of trans-hydrogen bond scalar couplings further allows a detailed characterization of hydrogen bonds [8,9]. We recently demonstrated that the 1JHN spin–spin coupling constant is increased by up to 1.6 Hz due to intramolecular hydrogen bond formation when compared to amino acid speciﬁc reference values observed in intrinsically disordered proteins . In addition, we did not detect any changes in 1JHN couplings due to changes in pH (pH 6.0–7.4) and temperature (5–25 °C) in the ⇑ Corresponding author at: German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany. E-mail address: [email protected]
(M. Zweckstetter). http://dx.doi.org/10.1016/j.jmr.2014.04.004 1090-7807/Ó 2014 Elsevier Inc. All rights reserved.
intrinsically disordered proteins Tau and a-synuclein. We also found that 1JHN couplings are increased in the 310-helix of the protein ubiquitin , whereas trans-hydrogen bond couplings are too small to be detected due to the speciﬁc geometry of hydrogen bonds in 310-helices. 1JHN can therefore be a useful observable to probe intramolecular hydrogen bonds in proteins . The coiled-coil motif is characterized by several a-helices wound into a superhelical structure and is one of the most widespread motifs in proteins [11–13]. In addition, coiled-coils provide a powerful model system to study the connection between primary sequence and 3D structure . Indispensable for folding of coiledcoils is a small autonomous folding unit termed the ‘‘trigger sequence’’ [15–17]. A well-studied example of coiled-coil is the GCN4 leucine zipper from the yeast transcription factor GCN4, which is formed by two identical peptide chains of 33 residue length . The trigger sequence of GCN4 is comprised by 16 residues at the C-terminus. Using NMR spectroscopy and molecular dynamics (MD) simulation, it was shown that GCN(16–31) populates a dynamic, partially helical conformation [18,19]. Moreover, mutagenesis and circular dichroism (CD) assigned a crucial role to R25 for stabilization of the conformation of the trigger sequence and the folding of the GCN4 coiled-coil . Here we investigated the possibility to detect transient intramolecular hydrogen bonds in the trigger sequence of GCN4 from 1 JHN scalar couplings and H/D exchange, with the GCN4 trigger sequence being at 15N and 13C natural abundance.
S. Xiang, M. Zweckstetter / Journal of Magnetic Resonance 243 (2014) 93–97
2. Results and discussion Previous studies have shown that GCN(16–31) is monomeric in solution, making it a viable model to study the folding of leucine zippers . CD showed that the peptide contained 15% helical conformation in 50 mM Na-phosphate buffer, pH 6.8 (Fig. 1). In line with the absence of a rigid helical conformation, secondary structure analysis on a single-residue level using NMR spectroscopy gave secondary structure propensity (SSP) scores  below 0.5, i.e. less than half the value expected for a rigid helix (Fig. 2(a)). Notably, SSP scores were higher in the middle part and decreased to near zero at the two ends. These results are consistent with previous studies, which revealed that GCN(16–31) populates a partially helical, dynamic conformation [18,19]. The aim of the current study was to obtain insight into the presence of backbone intramolecular hydrogen bonds in this partially helical, dynamic conformation. This is particularly challenging due to the low percentage of helical conformation (Fig. 2(a)). We therefore measured one-bond scalar couplings, 1JHN, between the protons and nitrogen atoms in the protein backbone using NMR spectroscopy. The identiﬁcation of intramolecular hydrogen bonds is then based on a decrease in the size of the 1JHN value, i.e. a more negative value as 1JHN is negative, when compared to a set of amino-acid speciﬁc random coil values that was previously determined for the intrinsically disordered proteins Tau and a-synuclein . Notably, our previous study had shown that the random coil 1 JHN values are highly robust against pH and temperature . NMR measurements were performed with a GCN(16–31) peptide concentration of 4 mM. Since the peptide is at 15N natural abundance, the effective GCN(16–31) concentration was 15 lM. Because of the high sensitivity of the 1H–15N BSD-IPAP-HSQC experiment, the coupling-induced peak splitting in the nitrogen dimension can be determined with high accuracy (0.05 Hz) even at a peptide concentration of 15 lM (Fig. 2(b) and Table 1). Fig. 2(c) shows the difference between experimentally observed 1 JHN and the amino-acid speciﬁc random coil values  – that is D1JHN secondary values – as a function of residue number of GCN(16–31). With the exception of residues 22 and 25, the magnitude of D1JHN was less than ±0.2 Hz. Taking into account that D1JHN values in rigid helices are approximately 1.0 Hz, the data suggest that most backbone amide protons in GCN(16–31) are involved in an intramolecular hydrogen bond less than 20% of the time. The most negative D1JHN value, D1JHN = 0.46 ± 0.09 Hz, was observed for R25. Assuming a value of 1.0 Hz when the helix is rigidly formed , this would correspond to an intramolecular hydrogen bond population of 46% for the amide proton of R25. A D1JHN
Fig. 1. CD spectra of GCN(16–31) in the far-UV region (185–260 nm) at TFE concentrations increasing from 0% to 40% (v/v). Spectra were obtained on a Chirascan spectropolarimeter (Applied Photophysics) using a 1-mm path cell at 25 °C. The peptide concentration was 0.2 mg/ml.
Fig. 2. Detection of intramolecular hydrogen bonds in GCN(16–31) using NMR spectroscopy. (a) Secondary structure propensity (SSP) scores  of wild-type (black) and the R25A variant (grey) of GCN(16–31) as a function of residue number. HN, N, Ca and Ha chemical shifts were obtained from two-dimensional HSQC experiments acquired on natural abundance peptides. (b) Superposition of a selected region of the in-phase (blue) and anti-phase (red) part of the BSD-IPAPHSQC of GCN(16–31). 1JHN values extracted from the spectra are indicated. (c) Deviation of 1JHN values observed in wild-type GCN(16–31) from residue-speciﬁc random coil values. Experimental 1JHN were measured using BSD-IPAP-HSQC experiments  with a data matrix of 256(N) 512(H) complex points. Experiments were repeated to estimate experimental errors. The two dashed lines indicated the estimated error range (±0.35 Hz) for D1JHN . (d) Deviation of 1JHN values observed in the R25A mutant of GCN(16–31) from residue-speciﬁc random coil values. The experimental error for the 5 C-terminal residues is large, as these residues had broad peaks in the mutant peptide. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)
S. Xiang, M. Zweckstetter / Journal of Magnetic Resonance 243 (2014) 93–97 Table 1 D1JHN and their errors values of GCN(16–31) peptide in buffer, 40% TFE, 4 M GdnHCl and R25A mutant in buffer.a Native GCN (16–31) 1
0.09 0.12 0.11 0.02 0.06 0.16 0.23 0.06 0.01 0.46 0.08 0.10 0.05 0.01 0.03 0.05
0.04 0.23 0.19 0.07 0.04 0.06 0.05 0.07 0.27 0.09 0.12 0.18 0.07 0.05 0.25 0.24
D JHN (Hz) N16 Y17 H18 L19 E20 N21 E22 V23 A24 R(A)25 L26 K27 K28 L29 V30 G31
40%TFE treated GCN(16–31) 1
0.01 0.21 0.26 0.14 0.38 0.05 0.85 0.25 0.46 1.02 0.58 0.61 0.12 0.46 0.16 0.03
0.07 0.00 0.45 0.10 0.12 0.12 0.06 0.10 0.33 0.08 0.03 0.03 0.13 0.06 0.07 0.05
D JHN (Hz)
4 M GdnHCl denatured GCN(16–31) 1
0.10 0.22 0.08 0.03 0.16 0.18 0.14 0.18 0.32 0.02 0.22 0.17 0.09 0.08 0.12 0.08
0.03 0.08 0.00 0.10 0.07 0.04 0.09 0.00 0.10 0.01 0.06 0.04 0.04 0.13 0.10 0.08
D JHN (Hz)
D1JHN (Hz) 0.04 0.28 0.04 0.19 0.21 0.12 0.07 0.14 0.23 0.30 0.01 0.21 0.10 0.06 0.13 0.10
Error (Hz) 0.06 0.05 0.16 0.10 0.24 0.06 0.07 0.06 0.03 0.01 0.02 0.25 0.26 0.29 0.28 0.31
a D1JHN values are the difference between experimental1JHN spin–spin coupling constants observed in GCN(16–31) and previously determined random-coil values . For further details, please see Section 4.
value of 0.46 ± 0.09 Hz also exceeds the previously deﬁned threshold of 0.35 Hz for identiﬁcation of intramolecular hydrogen bonds in rigid protons . Compared with an accuracy of the 1JHN measurement of 0.05 Hz, this threshold is quite conservative but takes into account any residual inﬂuence of pH, temperature and primary sequence on the 1JHN random coil values. Taken together our data demonstrate that the helical structure and the intramolecular backbone hydrogen bonds in the trigger sequence of GCN(16– 31) are highly transient. The amide proton of R25 however is involved in an intramolecular hydrogen bond on average 46% of the time, in line with its location at the center of the GCN(16– 31) trigger sequence. Next, we asked the question if the transient population of intramolecular hydrogen bonds is important for the presence of transient helical structure in GCN(16–31). To this end, we exposed GCN(16–31) to 4 M GdnHCl and determined secondary structure propensities and 1JHN values at single-residue level. Fig. 3 shows that the SSP scores of GdnHCl-treated GCN(16–31) were within the range of 0.2 to 0.1, indicating that GCN(16–31) was fully disordered. Therefore, no intramolecular hydrogen bond is expected to exist in this condition. Indeed, none of the D1JHN values in GdnHCl-treated GCN(16–31) exceeded the 0.35 Hz threshold (Fig. 3). Electrostatic interactions mediated by the side chain of R25 are essential for the stability of the GCN helix [21,22]. Mutation of R25 to alanine shifts GCN(16–31) towards the random coil state (Fig. 2(a)) [21,22]. We measured a natural-abundance 2D 1H–15N HSQC spectrum of R25A GCN(16–31). In this 2D 1H–15N HSQC, the cross peaks, which were observed for the ﬁve C-terminal residues in a 2D 1H–15N HSQC spectrum, were broadened when compared to the wild-type peptide. This suggests that the R25A mutation induces chemical exchange at the C-terminus of GCN(16–31). We then determined 13C chemical shifts and calculated the SSP score of R25A-GCN(16–31) (Fig. 2(a)). The analysis demonstrated that the overall helical content decreased by more than twofold. In addition, the 1JHN coupling of residue 25 decreased by 0.5 Hz and all D1JHN values were below 0.35 Hz (Table 1 and Fig. 2(d)). Assuming a D1JHN value of 1.0 Hz when a helix is rigidly formed , secondary 1JHN values indicate that the backbone amide protons are involved in intramolecular hydrogen bonds less than 35% of the time upon substitution of R25 by alanine. 2,2,2-Triﬂuoroethanol (TFE) is known to stabilize helical conformations. Indeed, stepwise addition of TFE from 0% to 40% increased the helical content in GCN(16–31) from 15% to 40% (Table 2 and
Fig. 3. (a) Secondary structure propensity (SSP) score of GCN(16–31) in buffer (dark grey), in 4 M GdnHCl (black) and 40% TFE (grey). (b) Secondary 1JNH values of GCN(16–31) in 4 M GdnHCl as a function of residue number. The two dashed lines indicated the estimated error range (±0.35 Hz) for D1JHN .
Table 2 Inﬂuence of TFE concentration on the secondary structure of GCN(16–31) based on the CD spectroscopy.
a-Helix (%) b-Sheet (%)
Fig. 1). Addition of TFE therefore is assumed to mimic later stages of the folding of leucine zippers – that is a stage that is closer to a rigid, fully formed helix as observed in the high-resolution 3D structures of leucine zippers. We then analyzed at single residue level the helical content and presence of intramolecular hydrogen bonds of GCN(16–31) in 40% TFE. In line with the stabilization of
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Fig. 4. Intramolecular hydrogen bonds in 40% TFE-treated GCN(16–31). Secondary 1 JHN as a function of residue number. The measurements were carried out in the same way as those for the native peptide. The two dashed lines indicated the estimated error range (±0.35 Hz) for D1JHN .
the helical structure, SSP scores were increased to about 0.8 for residues 18–27 (Fig. 3). The D1JHN values of GCN(16–31) in this condition are shown as a function of residue number in Fig. 4. For several residues negative D1JHN values were observed. In particular, the amide protons of E20, E22, R25, L26, K27 and K29 exceeded the threshold value of 0.35 Hz. Thus, the increase in helical content
is connected to an increase in the population of backbone intramolecular hydrogen bonds in GCN(16–31). To obtain further insight into the population of backbone intramolecular hydrogen bonds in GCN(16–31) we used hydrogen/deuterium (H/D) exchange. Previously, H/D exchange experiments had been carried out on a construct comprising the 32 C-terminal residues of GCN . GCN(1–32) forms a dimer, comprised of two rigid a-helices , and has well-formed intramolecular hydrogen bonds in line with H/D exchange . Compared to GCN(1–32), the amide protons in GCN(16–31) exchanged much faster with solvent precluding a quantitative analysis of their exchange rates: the backbone amide protons of all residues were exchanged to deuterium within 10 min in D2O (Fig. 5(a)). This suggests that the hydrogen bond of the amide proton of R25 is not stable enough to sufﬁciently slow down the H/D exchange process. In line with the transient nature of the R25 hydrogen bond, the secondary 1 JHN of R25 was only 0.46 Hz, i.e. about half the value expected in a stable intramolecular hydrogen bond (Fig. 2(c)). In the presence of 40% TFE, the central residues of GCN(16–31) exchanged three orders of magnitude faster than the corresponding part in the longer leucine zipper (Fig. 5(b) and (c)). Nevertheless, for four residues in the center of GCN(16–31), V23, A24, R25 and L26, NMR signals remained observable after 10 min exchange in D2O. Comparison with the D1JHN values observed in 40% TFE suggests that H/D exchange and 1JHN measurements are complimentary. Both observables indicate that the amide protons of R25 and L26 are involved in intramolecular hydrogen bonds in 40% TFE. H/D exchange further reveals slow solvent exchange for residues 23 and 24, while the experimental error of 1JHN of A24 is too large to make an unambiguous statement on the basis of D1JHN. We currently do not know the reason for the small D1JHN of V23. This might be caused by an unfavorable hydrogen bond geometry or dynamics in the helical structure, as for example observed for the C-terminal residue in the a-helix of ubiquitin . On the other hand, the 1JHN couplings of residues 22, 27 and 29 indicate that the backbone amide protons of these residues transiently form intramolecular hydrogen bonds (Fig. 4), but the stability is not sufﬁcient to be detected by H/D exchange measurements. 3. Conclusions In summary, we demonstrated that a transient intramolecular hydrogen bond is present in the partially helical folding nucleus of GCN(16–31). Our study also shows that 1JNH couplings are a sensitive tool for the detection of intramolecular hydrogen bonds in challenging biomolecules where the effective/useable concentration is low. This includes peptides at natural abundance but also uniformly labeled biomolecules that might be limited to low concentrations because of precipitation or aggregation. 4. Experimental
Fig. 5. Amide proton exchange in GCN(16–31). (a) 1D proton NMR spectra of GCN(16–31) in 50 mM Na-phosphate buffer, pH 6.8. In grey the reference spectrum recorded in water is shown. The black spectrum was recorded after 10 min in D2O. Resonance assignments are indicated. The remaining peak is from He1 of H18. (b) 1D proton spectra of 40% TFE-treated GCN(16–31). Color coding as in panel a. Only the central 4 residues, from 23 to 26, remained after 10 min in D2O. (c) Relative amide exchange rates of GCN as log(kex/kint) vs residue number. kex and kint are the observed and predicted intrinsic exchange rates, respectively. The solid line shows the data obtained from ﬁtting the time-dependent NMR signal decay of the trigger sequence GCN(16–31) in D2O. For comparison, relative exchange rates as observed in GCN(1–33) , which forms a stable helix, are shown (dashed line).
GCN(16–31) peptide and its R25A mutant were produced by standard Fmoc-solid-phase peptide synthesis using an ABI 433A synthesizer (Applied Biosystems). Peptides were synthesized with acetyl- and amide-protection groups at the N- and C-termini, respectively. Products were further puriﬁed by reversed-phase HPLC and the pure products were lyophilized. All peptides were dissolved in 50 mM sodium phosphate buffer, and were adjusted to pH 6.8. For NMR measurements, peptide concentrations were 4 mM. The peptide samples were diluted to 0.1 mg/ml in 50 mM sodium phosphate buffer, pH 6.8. CD spectra were recorded on an Applied Photophysics Chirascan instrument. A quartz cuvette
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with 1 mm path length was used to measure spectra at 0.5 nm interval over the 185–260 nm wavelength range at room temperature. The buffer spectrum were measured and subtracted from spectra of peptides. The secondary structure content was determined using the K2D2 program . NMR spectra were acquired at 278 K on Bruker 800 MHz spectrometers equipped with cryoprobes, and a 600 MHz spectrometer equipped with a room temperature probe. The resonance assignments of GCN(16–31) and its R25A mutant were transferred from a previous study , and veriﬁed by 2D TOCSY, DQF-COSY and natural abundance 2D 1H–13C HSQC spectra. DSS was used as internal reference. Secondary structure propensity (SSP) scores were calculated as described previously . 1JHN values were measured by BSD-IPAP-HSQC , in which long-range couplings such as 2JNHa, 3 JNHb, and 3JHNHa were refocused by REBURP pulses centered at 2.4 ppm and covering a bandwidth of 2.8 ppm. In-phase and antiphase spectra were recorded interleaved with 256(15N) 512(1H) complex points. To estimate experimental errors, BSD-IPAP-HSQC experiments were recorded four times for native GCN(16–31), twice for GCN(16–31) in 40% TFE, twice for 4 M GdnHCl treated GCN(16–31) and twice for the R25A mutant of GCN(16–31). The spectra were processed by nmrpipe. The peak positions were determined by the nonlinear ﬁtting routine nlinLS from the same suite of programs. Peptide samples in H2O were lyophilized and then re-dissolved in 100%, ice-cold D2O immediately before transfer into the NMR spectrometer. 1D proton experiments were measured every minute to monitor the decay of signals. The signal intensities of the amide protons were extracted at each time point and ﬁt to an exponential function, in order to extract exchange rates. The intrinsic exchange rates and the rates of observed in GCN(1–33) were taken from . Acknowledgment We thank Kerstin Overkamp for solid-phase synthesis. References  D.W. Bolen, G.D. Rose, Structure and energetics of the hydrogen-bonded backbone in protein folding, Annu. Rev. Biochem. 77 (2008) 339–362.  P. Neudecker, P. Robustelli, A. Cavalli, P. Walsh, P. Lundström, A. Zarrine-Afsar, S. Sharpe, M. Vendruscolo, L.E. Kay, Structure of an intermediate state in protein folding and aggregation, Science 336 (2012) 362–366.  W.W. Cleland, P.A. Frey, J.A. Gerlt, The low barrier hydrogen bond in enzymatic catalysis, J. Biol. Chem. 273 (1998) 25529–25532.  M.M.G. Krishna, L. Hoang, Y. Lin, S.W. Englander, Hydrogen exchange methods to study protein folding, Methods 34 (2004) 51–64.
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