Using MUSIC and CC(CO)NH for Backbone Assignment of Two Medium-Sized Proteins Not Fully Accessible to Standard 3D NMR

1. Introduction

2. Results and Discussion

2.1. CC(CO)NH Spectra and the Choice of Mixing Times

It was possible to identify the amino acid types from their aliphatic carbon shifts from all three spectra (mixing times of 22, 27 and 30 ms) in ubiquitin. Even though the average signal strengths appeared to be optimal with the shortest mixing time, longer mixing times have the advantage that they yield higher peak intensities for the remote carbons in the side chain, which often are most valuable for the assignment of the amino acid type. The protein concentrations of R17 and hNaa50p were only approximately 20% of that of ubiquitin and CC(CO)NH gets less sensitive with increasing molecular mass. However, it was sufficient to collect only one CC(CO)NH spectrum with the averaged long mixing time of 28 ms for these proteins, because most of the C^α and the C^β shifts were already obtained from standard experiments in advance.

Ten amino acid types can be directly identified by CC(CO)NH, whereas the remaining ten can be divided into three groups (Table 1). In ubiquitin, it was possible to assign almost all aliphatic carbons for all amino acid types. In R17 and especially hNaa50p, the information from CBCA(CO)NH combined with the results from CC(CO)NH could be used to identify or predict the most likely amino acid type (Figure 1). CBCA(CO)NH could determine glycine, alanine and the pair of serine and threonine, whereas CC(CO)NH was used to distinguish between the latter two amino acids. Amino acids with long aliphatic side chains were identified from the range of the C^γ (V), C^δ (L, P), C^γ and C^δ (I, R) and C^ε (K) chemical shifts.

Table 1. Amino acid type specificity of 3D CC(CO)NH using long isotropic mixing times of 27 (ubiquitin) or 28 ms. The table shows how many of the total number of an amino acid type could be identified in one small and two medium-sized proteins.

Table 1. Amino acid type specificity of 3D CC(CO)NH using long isotropic mixing times of 27 (ubiquitin) or 28 ms. The table shows how many of the total number of an amino acid type could be identified in one small and two medium-sized proteins.

Amino Acid	Ubiquitin	Detectable Signals a
		R17	hNaa50p
G	5/5	6/6	10/10
A	2/2	11/12	7/7
S	3/3	3/4	6/6
T	7/7	5/5	4/4
I	5/5	3/3	9/12
V	4/4	6/8	8/8
L	9/9	7/9	8/13
R	4/4	4/4	5/6
K	7/7	13/13	10/12
P	2/2	-	2/4
E/Q/M	12/12	17/21	6/14
N/D/F/Y/Cox	9/9	11/20	11/29
H/W/Cred	1/1	0/4	1/6

^a Amino acids that are predecessors of proline or of amino acids that have solvent or conformational exchanged NH-peaks were not included in the total number of that specific amino acid.

Figure 1. Amino acid type identification by the 3D CC(CO)NH experiment. The strips on the left and right side show spin systems of human ubiquitin and of hNaa50p, respectively. Even though CC(CO)NH is much less sensitive for larger proteins, a long mixing time allows for the observation of the remote carbons which are valuable for the assignment work. Crosses indicate chemical shifts that were determined from CBCA(CO)NH. Glutamate is shown as an example of the group that also comprises glutamine and methionine, which all have a similar chemical shift distribution.

Furthermore, the group of glutamine, glutamate and methionine was determined from the positioning of their C^γ shift. Many of the remaining seven amino acid types lacked signals apart from C^α, and could thus not be identified from CC(CO)NH. Table 1 shows how many amino acids could be detected out of the total amount of a specific type in the proteins.

2.2. MUSIC Spectra of Ubiquitin

Thirty different MUSIC experiments were tested on ubiquitin, and 27 of them yielded satisfactory results (Table 2). Three out of four proline-detecting experiments showed insufficient signal strengths and were not investigated further.

Other MUSIC techniques appeared to be rather insensitive, like A_ii+1, N_ii+1, and experiments (both versions) that detect threonine, leucine, arginine and lysine. Good signal strengths, on the other hand, were obtained from experiments detecting asparagine, aspartate, glutamine, glutamate, valine, isoleucine and the aromatic residues. Because most of these amino acid types are difficult to determine from CC(CO)NH, the corresponding experiments were the most promising for the assignment of the medium-sized proteins. Since the spectra from several variants can be superpositioned with each other, e.g., N_i+1, DNG_i+1, EQG_i+1 and NQ_i+1, it is possible to specifically assign most of the amino acids with acid and amide groups in their side chains.

Some MUSIC spectra contained artefacts which are in agreement with the previous reported ones, and that one should be aware of when running these experiments. The G_i+1 experiment, which is selective for CH₂-groups directly bound to an amide, leads to weak side chain NHD-peaks of asparagine and glutamine [13]. In the G_ii+1 spectrum on the other hand, which is selective for methylene groups bound to an amine, arginine side chain signals are observable [13]. However, these peaks are usually easily identified because of their spectral positions (asparagine and glutamine) and because of aliasing into the spectrum with sign inversion (arginine). Most glycine peaks in the DNG and EQG experiments are negative, and thus easily recognized. Some of the asparagine signals tend to be negative as well. Due to varying values of the ¹J_CC coupling constant, asparagine and aspartate cannot always be completely suppressed and might appear as weak negative artefacts in the EQG-spectra [15], but these can be identified from the superposition of the DNG- and EQG-spectra and the C^β chemical shift values. For the same reason, methyl signals belonging to valine, isoleucine or threonine might break through in the LA-spectra [15].

In the VIA experiments, valine tends to show a higher signal intensity than isoleucine because two γ-methyl groups contribute to the valine peaks [15]. In addition, these two amino acids can be easily distinguished from each other due to their differing C^β chemical shift values. Interestingly, in the FYH spectra the phenylalanines showed sign inversion compared to histidine and tyrosine. Since histidine has another C^β chemical shift range than the other two aromatics, this allowed for the direct identification of the three amino acid types in human ubiquitin.

Interestingly, even though both E24 and G53 are not observable in the ¹⁵N HSQC spectrum due to exchange-broadening, the two successors of these residues appear in the EQG_i+1 spectrum.

Table 2. Overview of 30 MUSIC experiments that were acquired on human ubiquitin.

Table 2. Overview of 30 MUSIC experiments that were acquired on human ubiquitin.

Variant a	Acquisition Time (h)	Signals	Artefacts	Comments
G_i+1 b	3	5/5	N c/Q c	weak artefact signals
G_ii+1 d	3	5/5	R c	artefacts are negative if aliased
A_i+1	3	2/2
A_ii+1	1.5	2/2		low sensitivity
S_i+1	4.5	3/3
S_ii+1	4.5	3/3
N_i+1	2	2/2
N_ii+1	8	2/2		low sensitivity
NQ_i+1	2	8/8
NQ_ii+1	2	8/8		One Q is negative
DNG_i+1	2	11/11		G and one N are negative
DNG_ii+1	2	12/12		G and one N are negative, N have lowest signal strengths
EQG_i+1	2	18/18	N	G and one Q are negative
EQG_ii+1	2	15/16	N	G and one Q are negative
TA_i+1	1.5	8/9		low sensitivity
TA_ii+1	1.5	9/9		low sensitivity
VIA_i+1	1.5	13/13		V has higher intensity than I
VIA_ii+1	1.5	13/13		V has higher intensity than I
LA_i+1	1.5	10/11	V/T	low sensitivity
LA_ii+1	1.5	9/11	T	low sensitivity
FYH_i+1	1	4/4		F are negative
FYH_ii+1	4	4/4		F are negative
R_i+1	1	4/4	G
R_ii+1	1	3/4	R c	low sensitivity
KR_i+1	6	10/11	G/S	low sensitivity
K_ii+1	6	4/7	G/R	low sensitivity, both positive and negative signals
P_i+1	2	2/2
P_i-1	2	0/2		low sensitivity
P_i+1np	2	0/2		low sensitivity
P_i-1np	4	0/2		low sensitivity

^a The i+1 variants detect the successor to a certain amino acid type, whereas the ii+1 versions identify the selected amino acid itself and, very weakly, its successor. ^b Amino acids (i+1) which have proline as successor or are at the C-terminus (E18, I36, P37 and G76), and amino acids (ii+1) that have solvent or conformational exchanged amide peaks (M1, E24 and G53) were removed from the total number of that specific amino acid. ^c Side chains. ^d Only the appearance of the residue itself (i) is annotated in the ii+1 experiments because the signal strengths of the successors are usually low and might be difficult to detect in medium-sized proteins. Some of the MUSIC methods were very sensitive, like G_i+1, G_ii+1, A_i+1 and S_i+1, but these amino acid types are easily identified by standard 3D techniques as well. However, these MUSIC experiments can be useful for diminishing assignment ambiguities in overlap regions.

2.3. MUSIC Spectra of R17 and hNaa50p

Twenty four out of the 27 MUSIC variants that yielded good results on ubiquitin were acquired on the almost 20 kDa protein hNaa50p (Table 3). Three of the least sensitive experiments (A_ii+1, KR_i+1 and NQ_ii+1) were not conducted.

Table 3. Overview of the MUSIC experiments that were acquired on the two-medium sized proteins.

Table 3. Overview of the MUSIC experiments that were acquired on the two-medium sized proteins.

Variant	hNaa50p		R17				Comments
	Acquisition Time (h)	Signals	Acquisition Time (h)		Signals
G_i+1 a	4	8/10	n.d. b		n.d.		side chain artefacts
G_ii+1 c	3	4/9	n.d.		n.d.
A_i+1 d	9	7/7	12.5		12/13
S_i+1	2	4/6	n.d.		n.d.
S_ii+1	5	0/5	n.d.		n.d.		low sensitivity
N_i+1	4	4/11	4		5/6
N_ii+1	8	1/11	n.d.		n.d.		low sensitivity
NQ_i+1	12	9/17	6		8/11
DNG_i+1	6	17/30	5		16/21		G and some N are negative, some G are missing (R17)
DNG_ii+1 d	10.5	8/28	17.5		17/20		E artefacts, G and some N are negative, D are strongest
EQG_i+1	5	8/22	5		20/24		N and D artefacts, also side chains, G are negative, Q are strongest
EQG_ii+1	10	9/23	8		14/24		N and D artefacts, also side chains, low G sensitivity (R17)
TA_i+1	3	0/14	n.d.		n.d.		low sensitivity
TA_ii+1	22	4/11	n.d.		n.d.		low sensitivity
VIA_i+1 d	22	23/27	10		22/23		V has higher intensity than I
VIA_ii+1 d	23.5	24/33	n.d.		n.d.		V has higher intensity than I
LA_i+1	1	12/20	n.d.		n.d.		low sensitivity
LA_ii+1	1	5/23	n.d.		n.d.		low sensitivity, even for A
FYH_i+1	10	6/18	8		7/8		all positive (R17), both positive and negative (hNaa50p)
FYH_ii+1 d	19.5	7/15	16		5/7		all positive (R17), both positive and negative (hNaa50p)
W_i+1 d	n.a. e	n.a.	10		1/1
W_ii+1 d	n.a.	n.a.	15.5		1/1
R_i+1	12	2/6	n.d.		n.d.
KR_i+1	1	0/18	n.d.		n.d.		low sensitivity, test spectrum only
K_ii+1	1	0/11	n.d.		n.d.		low sensitivity, test spectrum only
P_i+1	20	2/5	n.a.		n.a.

^a Amino acids (i+1) which have proline as successor or are at the C-terminus, and amino acids (ii+1) that have solvent or conformational exchanged amide peaks were removed from the total number of that specific amino acid. ^b Experiment not conducted ^c Only the appearance of the residue itself (i) is annotated in the ii+1 experiments since most of the successors are missing. ^d Shorter acquisition times might have been sufficient for these experiments. ^e Amino acid is missing from the protein sequence.

Several spectra showed small signal intensities, even at long acquisition times. The experiments which seem to be inappropriate for the use on this larger protein are: S_ii+1, N_ii+1 and those detecting threonine, arginine and lysine. Also, most of the ii+1 experiments, which are based upon 3D CBCANH, are significantly less sensitive than the i+1 version, which are based upon 3D CBCA(CO)NH, as expected from the sensitivity difference between these two 3D methods. However, the DNG, EQG and VIA ii+1 experiments resulted in rather good signal strengths.

Most of the artefacts and other features that were observed in the ubiquitin spectra were also present in the hNaa50p spectra. The only differences were that a few glutamate peaks appeared as artefacts in the DNG spectra, even though this was not expected since the DNG-sequence only includes one COSY step [15], and that the sign of the peaks in the FYH spectra could not be linked to the amino acid type.

Interestingly, the backbone assignment based on a repetition of the 3D standard experiments at high-field (800 MHz) were in agreement with the results obtained from CC(CO)NH and MUSIC at 600 MHz, and less than five additional peaks could be assigned using high-field NMR spectroscopy.

Only twelve of the MUSIC experiments, including those designed for tryptophan, were acquired on the 13 kDa protein R17 (Table 3), because of better signal resolution and strengths due to lower molecular mass making the assignment work less challenging than that of hNaa50p. Furthermore, the results from the MUSIC spectra on hNaa50p were taken into account so that the least sensitive methods were omitted.

Most of the selected MUSIC experiments worked very well on R17. Interestingly, the glycine signals were the least sensitive in the DNG and EQG experiments. Also, the peaks of the aspartate successors showed approximately the same signal strengths as the asparagine peaks in the DNG_ii+1 spectrum. That was surprising, because almost none of the successors were observable in any of the other ii+1 versions conducted on the medium-sized proteins. In the FYH experiments, all signals were positive. Thus, it was not possible to detect the amino acid identity directly from the spectrum. The presence of sign inversion in the spectra utilising L0S2-0 pulses (e.g., the FYH, DNG and EQG experiments) might indicate that the C^β and C^γ pulses were not optimal. However, all protein spectra were conducted with identical acquisition parameters and, for instance, all peaks in the R17 FYH spectra were positive whereas they showed sign inversion in the two other proteins. An example of how MUSIC experiments helped to identify residues in an overlap region is shown in Figure 2, whereas Table 4 views the amino acids that could be assigned to the primary sequences of R17 and hNaa50p.

3. Experimental

3.1. Sample Preparation

99% ¹³C (5 mg) and 98% ¹⁵N labelled human ubiquitin dissolved in 50 mM potassium phosphate buffer (600 μL, containing 10% D₂O), pH 5.8, was purchased from VLI Research Inc. (Malvern, PA, USA).

Uniformly ¹³C and ¹⁵N labelled R17 and hNaa50p were expressed and purified as previously described [21,26]. The concentration of both proteins was approximately 0.2 mM. R17 was dissolved in 20 mM sodium phosphate buffer (containing 10% D₂O) pH 6.8, 0.1 M NaCl, 1 mM DTT and 0.05% NaN₃ and hNaa50p in 50 mM sodium phosphate buffer (containing 10% D₂O) pH 7.4, 0.1 M NaCl and 1 mM acetylcoenzyme A.

Figure 2. The significance of MUSIC experiments in an overlap region of a medium-sized protein. Panel (A) shows parts of the CBCA(CO)NH-plane of R17 at 118.5 ppm (¹⁵N). This is the region with the most shift redundancy in the ¹⁵N HSQC spectrum. (B) The superposition of HSQC and three MUSIC spectra illustrates how the latter can assist the identification of spin partners in the CBCA(CO)NH spectrum. For instance, the VIA_i+1 peak at approximately 8.47 ppm (¹H) can easily be identified from the CBCA(CO)NH spectrum as an alanine successor (highlighted in a). The identity of the peaks and their predecessors are included in the HSQC spectrum.

3.2. NMR Experiments on Ubiquitin

The spectra were collected at 298 K and 600.13 MHz (¹H) on a Bruker Biospin AV600 spectrometer equipped with a superconducting actively shielded magnet. A 5 mm triple resonance (¹H, ¹³C, ¹⁵N) inverse cryogenic probehead with z-gradient coils and cold ¹H and ¹³C pre-amplifiers was used. The spectra were processed using Bruker Biospin’s TopSpin 1.3, and the resonance assignment was carried out in Cara [27].

The amide correlations were obtained from the ¹⁵N HSQC experiment [1,28]. Sequential backbone assignment was achieved from the spectra of the six standard 3D triple resonance experiments HNCA, HN(CO)CA, HNCO, HN(CA)CO, CBCA(CO)NH and HNCACB [29].

The 3D CC(CO)NH experiment [11,12] was acquired with isotropic mixing times of 22, 27 and 30 ms. The MUSIC experiments were collected using the pulses and parameters essentially as described in [13,14,15,16,17]; the pulse sequences and specific pulse shapes had been obtained from the Swiss Federal Institute of Technology, Zurich (ETHZ) [30].

Table 4. Overview of the amino acid sequences of hNaa50p (a) and R17 (b). Amino acids with assigned HN-peaks are highlighted. The columns denoted I and I-1 refer to the amino acid identity determined by the aliphatic chemical shifts and MUSIC experiments. Most unassigned residues are prone to exchange broadening. Note that the approach also works in stretches with repetitive amino acids (²¹EEEE²⁴ and ¹⁰⁵AAA¹⁰⁷ in R17).

Table 4. Overview of the amino acid sequences of hNaa50p (a) and R17 (b). Amino acids with assigned HN-peaks are highlighted. The columns denoted I and I-1 refer to the amino acid identity determined by the aliphatic chemical shifts and MUSIC experiments. Most unassigned residues are prone to exchange broadening. Note that the approach also works in stretches with repetitive amino acids (²¹EEEE²⁴ and ¹⁰⁵AAA¹⁰⁷ in R17).

a									b
Aa	I	I-1	Aa	I	I-1	Aa	I	I-1	Aa	I	I-1	Aa	I	I-1
M1			A58	A	G	Q114			M1			F60	FY	D
K2			V59	V	A	I115			A2	A		T61	T	FY
G3			C60			S116	ST	I	R3		A	V62		T
S4	ST	G	C61			N117		S	G4	G	R	H63		V
R5		S	R62			E118			Q5	EQ	G	K64		H
I6		R	V63	V	R	S119	S		R6		Q	D65	D	K
E7	E	I	D64		V	A120	A	S	L7		R	R66		D
L8		EQ	H65			I121	I	A	E8	EQ		V67	V	R
G9	G	L	S66	ST		D122	DN	I	E9			N68	N	V
D10		G	Q67		S	F123		DN	S10			D69		N
V11	V	DN	N68		Q	Y124			L11		S	V70	V	D
T12	T	V	Q69		N	R125			E12			C71		V
P13			K70		EQ	K126		R	Y13	FY	E	A72	A
H14			R71			F127	FY	K	Q14		FY	N73		A
N15		H	L72		R	G128	G		Q15		Q	G74	G	N
I16		DN	Y73		L	F129		G	F16		Q	E75	EQ	G
K17		I	I74			E130			V17	V		D76	D	E
Q18	EQ	K	M75		I	I131		EQ	A18	A	V	L77	L	D
L19	L	EQ	T76	ST		I132		I	N19			I78	I	L
K20		L	L77		T	E133		I	V20	V	N	K79
R21		K	G78	G		T134	T		E21	EQ	V	K80
L22		R	C79		G	K135		T	E22		E	N81	N	K
N23		L	L80			K136		K	E23	EQ		N82
Q24	EQ	DN	A81	A		N137		K	E24	EQ		H83
V25	V	Q	P82			Y138		N	A25	A		H84
I26	I	V	Y83		P	Y139		FY	W26	W	A	V85	V
F27			R84			K140			I27		W	E86	EQ
P28			R85			R141			N28	N	I	N87		E
V29	V		L86			I142	I	R	E29	EQ	N	I88		N
S30			G87	G	L	E143	EQ	I	K30		E	T89	T	I
Y31		S	I88		G	P144			M31		K	A90	A	T
N32			G89	G		A145	A	P	T32	T		K91		A
D33			T90	ST	G	D146		A	L33	L	T	M92		K
K34		DN	K91		T	A147	A	DN	V34	V	L	K93
F35		K	M92		K	H148	H	A	A35	A	V	G94	G	K
Y36			L93			V149	V		S36	S	A	L95		G
K37			N94		L	L150		V	E37	EQ	S	K96		L
D38			H95	H		Q151			D38	D	E	G97	G	K
V39	V		V96	V	H	K152			Y39	FY	D	K98		G
L40		V	L97		V	N153			G40	G	FY	V99	V	K
E41	EQ		N98		L	L154			D41	DN	G	S100	S	V
V42	V	EQ	I99			K155		L	T42	T	D	D101	D	S
G43		V	C100		I	V156	V	K	L43		T	L102		D
E44		G	E101	EQ		P157			A44	A	L	E103	EQ	L
L45		EQ	K102		EQ	S158	ST	P	A45	A	A	K104		E
A46	A	L	D103		K	G159	G	S	I46		A	A105	A	K
K47		A	G104	G	DN	Q160		G	Q47	EQ	I	A106	A	A
L48			T105	ST	G	N161	DN	EQ	G48	G	Q	A107	A	A
A49	A		F106		T	A162	A	N	L49		G	Q108	EQ	A
Y50		A	D107	DN		D163			L50		L	R109		Q
F51			N108			V164			K51			K110		R
N52			I109			Q165			K52		K	A111	A
D53			Y110			K166			H53	H	K	K112		A
I54			L111			T167			E54	EQ	H	L113		K
A55	A	I	H112			D168			A55	A	E	D114	D	L
V56	V	A	V113			N169			F56		A	E115	EQ	D
G57	G	V							E57	EQ	FY	N116	DN	E
									T58	T	E	S117	S	N
									D59	D	T	A118	A	S

4. Conclusions

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