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Stable Isotopes
Resonating with the Advancing Fieldof Biomolecular NMR
 
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Isotopic Labeling in Solid-state NMR of Proteins
Francesca M. Marassi, Ph.D.
The Burnham Institute • 10901 North Torrey Pines RoadLa Jolla, Caliornia 92037 USA
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Introduction
Isotopic labeling has played a crucialrole in the development o solid-stateNMR spectroscopy as a method ordetermining the structures o proteinsthat reside in membranes, aggregates,or other types o assemblies. With therealization that one-third o the proteinsequences in a genome correspond tointrinsic membrane proteins, there has been a surge o interest inthe urther development and routine application o solid-state NMRto membrane proteins. Oriented sample methods are particularlywell-suited or membrane proteins because o the highly asymmetricproperties o phospholipid bilayers.
1
The availability o a wide variety o labeled amino acids, carbon andnitrogen sources, as well as bacterial growth media are essentialor the preparation o samples o isotopically labeled proteins.There are three main approaches to isotopic labeling o proteins:specic, selective, and uniorm labeling, and all are used in solid-state NMR studies o proteins. Most o the eort has been ocusedon structure determination; however, these same samples can beused to characterize the dynamics o backbone and side chain sites.An important advantage o NMR spectroscopy over other methodso studying proteins is its ability to describe both the structure andmotions o individual sites.
Specifc vs. Selective Isotopic Labeling
Specic isotopic labeling reers to the placement o a single label(e.g.,
2
H,
13
C, or
15
N) in a specied location in a polypeptide.Nearly always, specic labeling is accomplished by incorporating alabeled amino acid through chemical synthesis, which restricts thisapproach to relatively small polypeptides, although recent advancesin synthetic methods have made it possible to specically label an81-residue membrane protein or NMR studies.
2
In avorable cases,specic labeling can be done biosynthetically i the expressed proteinsequence contains only a single copy o an amino acid, and, in somecases, it can be arranged by site-directed mutagenesis. Generally,specic labeling is employed in situations where the spectroscopicexperiments provide very limited or no opportunities or resolutionamong sites. In the initial stages o development o solid-state NMRo proteins, isotopic labeling had to carry most o the burden o theexperiments. For example, a single
15
N label at a specic site providesthe sensitivity needed to observe signals, distinguishes the signalsrom background, assigns the resonance to a specic site in theprotein, and provides the spin-interactions that are the sources o therequencies and other spectroscopic parameters that are measured.The initial application o solid-state NMR to an aligned protein wasperormed by biosynthetically labeling the indole nitrogen o thesole tryptophan sidechain in the protein with
15
N.
3
This enabled the
15
N NMR spectrum o an unoriented powder sample to be used toveriy that the site did not undergo motional averaging on the NMRtimescales and, by comparison, to demonstrate the dramatic spectralsimplications that enabled the direct measurement o requenciesand splittings that provide orientation constraints as input orstructure determination. The most comprehensive study perormedwith specic labeling through chemical synthesis is the structuredetermination o the ion channel peptide gramicidin in phospholipidsbilayers.
4
Nevertheless, while specic labeling was crucial in the initial stages odevelopment, and remains useul as a tool, it is inecient orcomprehensive studies because o the requirement or the separatepreparation o many samples. As the instrumentation and experimentalmethods have become more powerul, it has become easible toutilize samples where the proteins are labeled in multiple sites.Selective labeling reers to the biosynthetic incorporation o a singletype o labeled amino acid with all o the others unlabeled. Forexample, i a protein has eleven leucines, then only those elevenresidues will be labeled, and the labels provide sensitivity and improveresolution. As with specic labeling, the low natural background, sodetrimental to the sensitivity o
13
C and
15
N, became an advantagebecause signals rom only the labeled sites are observed in thespectra. Selective labeling is widely used to assist resonanceassignments in both solution NMR and solid-state NMR experiments;however, it has special signicance in solid-state NMR o alignedsamples where the mapping o protein structure onto the patternso resonances in the spectra rom selectively labeled samples servesas a method to simultaneously assign resonances and determinestructure.
5
 Combinations o specic and selective labeling are essential or thesolid-state NMR methods used to measure specic distances betweenstrategically placed pairs o nuclei in unoriented samples o proteins,including rotational resonance
6
and REDOR.
7
 
Uniorm Labeling
Uniorm labeling reers to biosynthetic labeling o all carbon,nitrogen, or hydrogen sites with stable isotopes. Uniorm labelingo proteins with
15
N is particularly convenient because o thestrategic locations o nitrogens in the backbone and the absence ohomonuclear couplings due to the intervening carbons.
8
It is alsopossible to replace all o the carbons with
13
C, although in this casethe spectroscopy has to deal with the network o couplings amongbonded carbons. Similarly, all o the hydrogens can be replaced withdeuterons in order to attenuate the dipolar couplings among thehydrogens that oten present complications and diculties in theexperiments.Until recently, the vast majority o solid-state NMR studies have beenperormed with specic or selective isotopic labeling; however, thiswas accomplished at a considerable cost in fexibility. For example,when many resonances are resolved in a spectrum rom a uniormlylabeled protein, assignments can be readily made by comparisonswith spectra rom selectively labeled samples. But this is not possiblei adequate resolution is present only in spectra o selectively labeledsamples. Uniorm labeling has the eect o shiting the experimentalburdens rom the isotopic labeling to the spectroscopy, which isundamentally more powerul and fexible and now capable oyielding completely resolved spectra o membrane proteins inphospholipids bilayers.
9
Uniorm labeling with
13
C and/or
15
N is readily accomplished bygrowing bacteria in media containing
13
C labeled glucose and/or
15
NH
4
Cl and opened up many avenues or NMR studies o proteins.Concurrent advances in molecular biology meant that a wide varietyo prokaryotic and eukaryotic proteins could be expressed in highyields in
E. coli 
grown on appropriately labeled media. This startedwith systematic methods or making resonance assignments insolution NMR spectra o proteins
10
and enabled the acquisition othe extensive chemical shit data that contributes to the constraintsavailable or structure determination. It now includes magic anglesample spinning solid-state NMR o insoluble aggregates
11, 12
andpolycrystalline proteins.
13
In some cases, there are advantages torandom ractional labeling with
13
C.
 
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 sigma-aldrich.com/isotec 
Combining Uniorm and Selective Labeling
A common use o selectively labeled samples in both solution NMRand solid-state NMR studies o proteins is to compare spectra romuniormly and selectively labeled samples. This enables the resonancesrom one type o amino acid to be identied by inspection. Althoughlimited in scope, this inormation is valuable because it is a reliableway o resolving ambiguities in alternative assignment schemes, andwhile isotopic scrambling occurs in some cases, it is generally readilyrecognized and can be taken into account.The comparison o two-dimensional solid-state NMR spectra o uni-ormly and selectively
15
N labeled samples is the basis or an approachto simultaneous resonance assignment and three-dimensional struc-ture determination o membrane proteins in lipid bilayers. This methodutilizes proteins aligned in the eld o the NMR magnet and relies onPisa Wheels (polarity index slant angle) to rst, obtain shotgun-styleresonance assignments and structure assembly o isolatedpolypeptide segments o the protein, and second, to assemble thesegments in their correct order and obtain the ull three-dimensionalstructure.
5
Pisa Wheels are circular patterns o resonances in two-dimensional PISEMA (polarization inversion spin exchange at themagic angle) spectra that mirror the helical wheels o membraneprotein helices in oriented bilayers.
14, 15
This approach short-circuitsthe laborious process o obtaining complete sequential assignments,greatly accelerating the process o structure determination.
Backbone and Side-chain Dynamics
Deuteration can be used in two distinct ways.
2
H can be a veryeective specic or selective label, as more commonly employedwith
13
C or
15
N, and the quadrupolar interaction provided by this spinone nucleus can be used in solid-state NMR experiments to describeboth aliphatic and aromatic side-chain dynamics.
16
The second wayor deuteration to be used in solid-state NMR experiments is similarto the approach used in solution NMR o larger proteins becauseo the benecial eects o dilution o the larger number o nearbyhydrogens in a protein.
Future Prospects
Based on the success o isotopic labeling in solution NMR
17
andin solid-state NMR as summarized briefy in this article, one canlook orward to the implementation o even more elegant isotopiclabeling schemes that will complement the development oinstrumentation and experimental methods or NMR studieso proteins.
Reerences
1.
Opella S. J., and Marassi F. M. (2004). Structure determination o membraneproteins by NMR spectroscopy.
Chem. Rev.
 
104
, 3587-3606.
.
Kochendoerer G. G., Jones D. H., Lee S., Oblatt-Montal M., Opella S. J., andMontal M. (2004). Functional characterization and NMR spectroscopy on ull-length Vpu rom HIV-1 prepared by total chemical synthesis.
 J. Am. Chem.Soc.
 
16
, 2439-2446.
.
Cross T. A., and Opella S. J. (1983). Protein structure by solid-state NMR.
 J. Am. Chem. Soc.
105
, 306-308.
4.
Ketchem R. R., Hu W., and Cross T. A. (1993). High-resolution conormationo gramicidin A in a lipid bilayer by solid-state NMR.
Science
61
, 1457-1460.
5.
Marassi F. M., and Opella S. J. (2003). Simultaneous assignment and structuredetermination o a membrane protein rom NMR orientational restraints.
Protein Sci.
 
1
, 403-411.
6.
Spencer R.G., Halverson K.J., Auger M, McDermott A.E., Griin G.R., andLansbury P.T. Jr (1991) An unusual peptide conormation may precipitateamyloid ormation in Alzheimer’s disease: Application o solid-state NMR to thedetermination o protein secondary structure.
Biochemistry 
 
0
, 10382-10387.
7.
Hing A.W. and Schaeer J. (1993) Two-dimensional rotational-echo doubleresonance o Val
1
-[1-
13
C]Gly
2
-[
15
N]Ala
3
-Gramicidin A in multilamellardimyristolphosphatidylcholine dispersions.
Biochemistry 
 

, 7593-7604.
8.
Cross T. A., DiVerdi J. A., and Opella S. J. (1982). Strategy or nitrogen NMRanalysis o biopolymers.
 J. Am. Chem. Soc.
 
104
,1759-1761.
9.
Marassi F. M., Ramamoorthy A., and Opella S. J. (1997). Complete resolutiono the solid-state NMR spectrum o a uniormly
15
N-labeled membrane protein inphospholipid bilayers.
Proc. Natl. Acad. Sci. USA
 
94
, 8551-8556.
10.
Ikura M., Kay L. E., and Bax A. (1990). A novel approach or sequentialassignment o
1
H,
13
C, and
15
N spectra o proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin.
Biochemistry 
 
9
, 4659-4667.
11.
Tycko R. (2003). Insights into the amyloid olding problem rom solid-stateNMR.
Biochemistry 
 
4
, 3151-3159.
1.
Jaroniec C. P., MacPhee C. E., Bajaj V. S., McMahon M. T., Dobson C. M.,and Griin R. G. (2004). High-resolution molecular structure o a peptide inan amyloid ibril determined by magic angle spinning NMR spectroscopy.
Proc.Natl. Acad. Sci. USA
 
101
, 711-716.
1.
Castellani F., van Rossum B., Diehl A., Schubert M., Rehbein K., and OschkinatH. (2002). Structure o a protein determined by solid-state magic-angle-spinning NMR spectroscopy.
Nature
 
40
, 98-102.
14.
Marassi F. M., and Opella S. J. (2000). A solid-state NMR index o helicalmembrane protein structure and topology.
 J. Magn. Reson.
 
144
, 150-155.
15.
Wang J., Denny J., Tian C., Kim S., Mo Y., Kovacs F., Song Z., NishimuraK., Gan Z., Fu R., et al. (2000). Imaging membrane protein helical wheels.
  J. Magn. Reson.
 
144
, 162-167.
16.
Opella S. J. (1986). Protein dynamics by solid-state nuclear magnetic resonance.
Methods Enzymol.
 
11
, 327-361.
17.
Gardner K. H., and Kay L. E. (1998). The use o
2
H,
13
C,
15
N multidimensionalNMR to study the structure and dynamics o proteins.
 Annu. Rev. Biophys.Biomol. Struct.
 
7
, 357-406.
Products for Minimal Media
I   s  o t  o pi   c L  a b  el  i  n g
 
Name Isotopic Purity Cat. No.
Ammonium-
15
N chloride98 atom %
15
N
299251-250MG299251-1G299251-20G
Ammonium-
15
N,d
4
deuteroxidesolution, ~ 3 N in D
2
O99 atom %
15
N98 atom % D
594091
Ammonium-
15
N hydroxidesolution, ~ 3 N in H
2
O98 atom %
15
N
488011-5G488011-10G
Ammonium-
15
N
2
sulate98 atom %
15
N
299286-250MG299286-1G299286-10G299286-20G
Deuterium oxide99.9 atom % D
151882-10G151882-25G151882-100G151882-125G151882-250G151882-500G151882-1KG151882-1.107KG
Deuterium oxide99.8 atom % D
617385-1KG617385-1.107KG
Deuterium oxide99 atom % D
435767-25G435767-100G435767-1KG
Deuterium oxide70 atom % D
613428
Name Isotopic Purity Cat. No.
d
-
Glucose-C-d
7
97-99 atom % D
552003-100MG552003-1G552003-10G
d
-Glucose-d
12
97-99 atom % D
616338-250MG
d
-Glucose-1-
13
C99 atom %
13
C
297046-250MG297046-1G297046-10G
d
-Glucose-
13
C
6
99 atom %
13
C
389374-100MG389374-250MG389374-1G389374-2G389374-3G389374-10G
d
-Glucose-
13
C
6
,C-d
7
99 atom %
13
C97-99 atom % D
552151-500MG552151-1G552151-5G
Glycerol-d
8
98 atom % D
447498-1G447498-5G
Glycerol-1,3-
13
C
2
99 atom %
13
C
492639-250MG
Glycerol-
13
C
3
99 atom %
13
C
489476-500MG
Glycerol-
13
C
3
, d
8
99 atom %
13
C98 atom % D
8
669024-500MG
Sodium pyruvate-3-
13
C99 atom %
13
C
490733-250MG
of 00

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