Supplementary Information
1
Supplementary Figures
Supplementary Figure S1. Peak-picking of the HSQC spectrum of human ubiquitin in Bruker
TopSpin 3.2 for setup of the F1F2-selective experiments. The numbers are temporary indices
before assignment. After peak-picking, 72 F1F2-selective (HNCA)NH experiments were set
up and recorded, each of which selectively probes one of the amide resonances shown here.
2
Supplementary Figure S2. Establishing the amino-acid connectivity along the primary
sequence of ubiquitin by the F1F2-selective (HNCA)NH experiment. As shown in Figure 3 of
the main text, each F1F2-selective (HNCA)NH experiments yields a connectivity between
three amino acids (i−1), (i) and (i+1) (hereafter referred to as: triplet). These triplets are
shown here in orange and aligned in such a way that a sequential connection along the
primary sequence is established due to the overlap between two triplets (see main text for
details). The numbers of the triplets refer to the peaks picked as shown in Supporting Figure
S1. Additional information about the amino acid type from the amino-acid type-selective
experiments (“MUSIC”) is shown in blue. Combining the information from amino-acid typeselective experiments and the connectivity between the triplets, one can fill in the primary
sequence of ubiquitin into the missing gaps (yellow). The connectivity breaks at proline
residues (Pro19, Pro38 and Pro39) or residues that are not observed in the HSQC spectrum due
to conformational exchange broadening (Glu24 and Gly53). Thus, the assignment is obtained
separately in 7 fragments. Note that only a few of the MUSIC experiments were used to
complete the assignment. The remaining experiments can be used for validation. X:
connectivity not unambiguous or end of cluster (N- or C- terminus, proline residue or
conformational exchange peak).
3
Supplementary Figure S3. F1F2-selective NOESY spectra of human FABP4 taken at
temperatures of 298 K (upper panel) and 287 K (lower panel).
4
Supplementary Figure S4. F1F2-selective R1ρ relaxation dispersion experiments on human
FABP4. A) An HSQC spectrum of FABP4 indicating examples of resonances for which
relaxation dispersion experiments were conducted. B) R1ρ relaxation dispersion profiles
obtained for Gly35 and Ile63 at a temperature of 298 K using the pulse sequence of Figure 5.
5
Supplementary Figure S5. Algorithm for automated backbone assignment of proteins from 2D
(HNCA)NH spectra exclusively. Additional information from amino-acid type-selective experiments
can be incorporated at the step “read amino acid information”.
6
Supplementary Tables
Supplementary Table S1. Time domain and spectral width of the F1F2-selective experiments
discussed here.
F1F2-selective experiment
Complex points
(direct / indirect dim.)
Sweep width [ppm]
(direct / indirect dim.)
(HNCA)NH
1024 / 24
14.0 / 34.0
NOESY-[1H, 15N]-HSQC
1024 / 24
12.0 / 40.0
NOESY-[1H, 13C]-HSQC
1024 / 48
12.0 / 40.0
[15N]-R1ρ relaxation dispersion
1024 / 8
12.0 / 1.2
7
Supplementary Table S2. Relative sensitivity of the experiments reported in this study as
compared to a sensitivity improved HSQC experiment taken under equivalent conditions
(number of scans, the time domain, sweep width, receiver gain, relaxation delay etc.). The
downfield amide resonance of Ile13 of ubiquitin was used for intensity quantification. In the
NOESY experiment, the peak corresponding to auto-relaxation was quantified (mixing time:
20 ms). In the case of the R1ρ relaxation dispersion experiment, the spin-lock was omitted.
Experiment
Intensity ratio [Iexperiment / IHSQC]
(HNCA)NH
3%
NOESY-[1H, 15N]-HSQC
43 %
[15N]-R1ρ relaxation dispersion
72 %
8
Supplementary Methods
De novo assignment from F1F2-selective (HNCA)NH
Without any prior assignment information on ubiquitin, we quickly connected the amino acid
triplets obtained from the F1F2-selective (HNCA)NH spectra to form seven larger fragments
in ubiquitin, thereby establishing sequential connections between all the amide resonances
observed in the HSQC spectrum (Supplementary Figures S1 and S2). To assign these
fragments to the primary sequence of ubiquitin, we obtained 2D amino acid-type selective
spectra (Schubert et al. 2000; Schubert et al. 2005; Schubert et al. 2001a; Schubert et al.
2001b; Schubert et al. 2001c; Schubert et al. 1999). By simple overlay with the HSQC
spectrum, these experiments showed quickly which of the amide cross-peaks in the HSQC
spectrum of ubiquitin were glycine, alanine, arginine, aromatic (Phe/Tyr/His), glutamine,
serine, aspartate, asparagine and proline-preceding (Pro−1) residues (Supplementary Figure
S2). The combined information of the F1F2-selective (HNCA)NH experiments and the amino
acid type selective experiments allowed unambiguous complete assignment of all observed
peaks in the HSQC spectrum of ubiquitin (Supplementary Figures S1 and S2).
We note that there is a convenient degeneracy in the F1F2-selective (HNCA)NH dataset,
because a given residue u “sees” two residues v and w, but u is also seen from w and from v.
Thus, if the experimental data connecting u → v somewhat ambiguous (e.g. due to peak
overlap or low signal-to-noise ratio), one can check if u is seen from v to resolve the
ambiguity. Moreover, since not all of the 16 amino-acid-type selective experiments had to be
used to obtain complete assignment of ubiquitin, these data allow an independent consistency
check of the obtained assignment. Together with the arguments presented in the main text,
this establishes that the F1F2-selective experiments can be used to backbone assignment of
proteins de novo.
Amino acid-type selective experiments
All experiments were acquired using the default Bruker pulse programs available in TopSpin
3.2 acquisition software: music_de_3d (Asp/Glu), music_fhyw_3d (Phe/His/Tyr/Trp),
music_gly_3d (Gly), music_ser_3d (Ser), music_ile_3d (Ile), music_qn_3d (Asn/Gln),
music_kr_3d (Lys/Arg), music_lavia_3d (Leu/Ala; Val/Ile/Ala), music_tavi3d (Val/Ile;
Thr/Ala) music_pro_1_3d (Pro−1). In total, 16 datasets were recorded with different labeling
schemes. All of these spectra were recorded as 2D experiments by setting the number of data
points to be acquired in the F1 dimension to 1. Note that only a few of the spectra were
actually used to establish the assignment (Supplementary Figure S2; blue rows labeled
“MUSIC”). Accordingly, the additional spectra are beneficial for validation of the
assignment.
9
New pulse programs
The pulse program and parameters files (in Bruker format) are available on the homepage of
our laboratory: http://www.moleng.kyoto-u.ac.jp/~moleng_01/
10
F1F2-selective (HNCA)NH Pulse Program Parameters
The parameters used were as follows. Most of these parameters were adopted from the
original publication of the HN(CA)NNH experiment (Weisemann et al. 1993) or the Bruker
file hncannhgp3d.2.
φ1
y
φ2
x
φ3
y, −y
φ4
2(x) 2(−x)
φ5
8(x), 8(−x)
φ6
4(x), 4(−x)
φ7
4(−y), 4(y)
φrec
x, −x, −x, x, −x, x, x, −x
G1
25 G/cm
G2
25 G/cm
G3
11.5 G/cm
G4
40 G/cm
G5
4.05 G/cm
11
F1F2-selective NOESY-[1H, X]-HSQC Pulse Program Parameters
The parameters used were as follows. Most of these parameters were adopted from the
sensitivity-improved NOESY-HSQC experiment or the Bruker file noesyhsqcf3gpsi3d.
φ1
−y
φ2
2(−x) 2(x)
φ3
4(x) 4(−x)
φ4
x
φ5
x (−x)
φ6
8(x) 8(−x)
φ7
8(x) 8(−x)
φ8
8(y) 8(−y)
φrec
x, −x, −x, x, −x, x, x,
−x
Δ
1 / 4JHX
G1
25 G/cm
G2
15 G/cm
G3
25 G/cm
G4
40 G/cm
G5
2.5 G/cm
G6
−1 G/cm
G7
4.05 G/cm
12
F1F2-selective 15N relaxation dispersion Pulse Program Parameters
The parameters used were as follows.
φ1
2(y) 2(−y)
φ2
x, −x
φ3
y
φ4
−y
φ5
x
φ6
−y
φrec
x, −x, −x, x
G1
3 G/cm
G2
15 G/cm
G3
−30 G/cm
G4
40 G/cm
G5
2.5 G/cm
G6
−1 G/cm
G7
4.05 G/cm
13