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Protein global fold determination using site-directedspin and isotope labeling

VADIM GAPONENKO,1 JACK W. HOWARTH,1 LINDA COLUMBUS,2

GENEVIEVE GASMI-SEABROOK,1 JIE YUAN,1 WAYNE L. HUBBELL,2

and PAUL R. ROSEVEAR1Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine,Cincinnati, Ohio 45267

2Departments of Chemistry and Biochemistry and the Jules Stein Eye Institute, University of California,Los Angeles, California 90095

~Received August 2, 1999;Final Revision October 28, 1999;Accepted November 25, 1999!

Abstract

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizessite-directed spin labeling~SDSL! in combination with isotope enrichment to determine long-range distance restraintsbetween amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxationrates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have beendemonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase,~H102C! and~H102A0Q15C!, were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue ofeach. Measurement of amide1H longitudinal relaxation times, in both the oxidized and reduced states, allowed thedetermination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from thefrequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 Å werecalculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide1Hcorrelation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structurecalculations. Using nitroxide to amide1H distances as long-range restraints and known secondary structure restraints,barnase global folds were calculated having backbone RMSDs,3 Å from the crystal structure. This approach makesit possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.

Keywords: barnase; EPR; NMR; paramagnetic enhancement; protein global fold; site-directed spin labeling

With the rapid increase in the number of newly mapped genes, thedevelopment of techniques for rapid determination of function isbecoming more important. Up to 40% of genes identified in com-pleted genomes represent novel proteins with unknown functionnot readily identified by homologous sequence comparison~Delsenyet al., 1997; Bork & Koonin, 1998!. Protein global folds can beused to suggest possible functions for these unknown proteins. Ithas previously been demonstrated that protein global fold struc-tures are sufficient to identify active site residues using modern

structure prediction algorithms~Fetrow & Skolnick, 1998! andSAR-type approaches~Shuker et al., 1996!.

The use of conventional techniques for protein structure deter-mination is often labor intensive and inefficient. Also, solutionstructure determination becomes problematic at larger molecularweights. With increasing molecular weight, the number of resolv-able interproton NOEs decreases and may yield an insufficientnumber of restraints for protein structure elucidation. To alleviatethis problem, long-range HN-HN NOEs obtained from perdeuter-ated proteins~Pachter et al., 1992; Grzesiek et al., 1995; Venterset al., 1995; Briercheck & Rule, 1998! as well as HN-HN, HN-methyl, and methyl-methyl NOEs obtained from highly deuterated0methyl-protonated proteins~Gardner et al., 1997! have been used.However, as the molecular weight of the system increases, thisapproach alone may prove insufficient.

Novel approaches that minimize the time required to determinea global fold and decrease the dependence on NOE restraints arenecessary to increase the efficiency of NMR in identification offunction of newly discovered proteins. Currently, protein structuredetermination heavily relies on the dipolar interactions between

Reprint requests to: Paul R. Rosevear, Department of Molecular Genet-ics, Biochemistry, and Microbiology, University of Cincinnati, College ofMedicine, 2015A Medical Sciences Building, 231 Bethesda Avenue, Cin-cinnati, Ohio 45267; e-mail: [email protected].

Abbreviations:DTT, dithiothreitol; EPR, electron paramagnetic reso-nance; HSQC, heteronuclear single quantum coherence; IPTG, isopropyl-b-d-thiogalactopyranoside; MTSSL, 1-oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-~methyl!methanethiosulfonate spin label; NOE, nuclear Overhauser effect;NOESY, NOE spectroscopy; RMSD, root-mean-square deviation; SDS,sodium dodecyl sulfate; S0N, signal-to-noise ratio.

Protein Science~2000!, 9:302–309. Cambridge University Press. Printed in the USA.Copyright © 2000 The Protein Society

302

protons, which are restricted to distances under 6 Å. Nitroxide spinlabels can provide long-range distance restraints up to 35 Å thatfacilitate global fold determination.

Unpaired electrons of spin labels, such as the nitroxide spinlabel MTSSL produce local fluctuating magnetic fields, that caninfluence the spin-lattice relaxation times of magnetic nuclei in adistance dependent manner. It was first suggested in 1967~Mc-Connell, 1967; Sternlicht & Wheeler, 1967! that paramagneticrelaxation times can be used for distance calculations and for ob-taining solution structural information on biomolecules. In one ofthe first studies, one-dimensional NMR difference spectroscopywas used to measure selective broadening of specific resonancesinduced by the presence of a nitroxide spin label covalently at-tached to hen egg white lysozyme~Schmidt & Kuntz, 1984!. Dis-tances between an unpaired electron on the spin label and affectedprotons were estimated using ther 26 distance dependence of themagnitude of paramagnetic line broadening of individual reso-nances in difference spectra of the oxidized~paramagnetic! andreduced~diamagnetic! nitroxide labeled hen egg white lysozyme~Schmidt & Kuntz, 1984!. The structural information obtained inthis research was not validated against the known molecular struc-ture of hen white lysozyme~Blake & Swan, 1971!. A similarmethodology was employed by Anglister et al.~1984! to structur-ally characterize a Fab fragment of a monoclonal antibody directedagainst a nitroxide spin labeled hapten. Two-dimensional J-correlated1H NMR spectra of nitroxide spin labeled bovine pancreatic tryp-sin inhibitor in the oxidized and reduced states of the spin labelallowed them to conclude that the spin label affects relaxationparameters of protons within;15 Å from the unpaired electron~Kosen et al., 1986!. These studies suggested the feasibility ofaccumulating a sufficient number of intramolecular distances fordetermination of protein solution structures. Kuliopulos et al.~1987!were able to study the position of a spin labeled steroid onD-5-3-ketosteroid isomerase by calculating nitroxide electron-protondistances from measured longitudinal and transverse relaxationrates.

More recently, Gillespie and Shortle~1997! used the paramag-netic enhancement from nitroxide spin labels to perform structuralanalysis on a fragment of staphylococcal nuclease and model thedenatured state. Spin labels were introduced at 14 unique positionsalong the polypeptide chain. The amide proton longitudinal andtransverse relaxation rates were measured and spin label-protondistances up to 25 Å estimated using the Solomon–Bloembergenequations~Solomon, 1955!. Distance restraints were used to pre-dict the global topology of the denatured state.

In the present study, we demonstrate the use of site-directed spinlabeling and isotope enrichment in determination of the globaltopology of barnase, a ribonuclease secreted byBacilus amy-loliquifacience. Several crystal structures of barnase are available~Mauguen et al., 1982; Baudet & Janin, 1991; Serrano et al., 1992;Buckle et al., 1993, 1994; Guillet et al., 1993; Schreiber & Fersht,1993; Schreiber et al., 1994!. In addition, NMR backbone assign-ments and the NMR solution structure of barnase have been pub-lished ~Bycroft et al., 1991!. The availability of both X-ray andNMR structures, as well as NMR assignments, makes barnase anexcellent model to access the quality of global fold structurescalculated using paramagnetic restraints. Site-directed spin label-ing was used to introduce a single nitroxide side chain at eitherHis102 or Gln15. Paramagnetic contributions to the amide protonlongitudinal relaxation times were then determined. Correlationtimes for the proton-electron vectors were estimated from the fre-

quency dependence of the paramagnetic relaxation times measuredat 800 and 600 MHz. Using the individual correlation times andthe paramagnetic contribution to the relaxation rate, nitroxideto amide proton distances were calculated using Solomon–Bloembergen equations~Solomon, 1955!. Distances, as well asknown restraints for the secondary structure, were used in X-PLORto calculate a family of global fold structures. A backbone atomRMSD of 2.9 Å for the average global fold structure from theknown crystal structure was determined. In the absence of second-ary structure obtained by either NMR or secondary structureprediction, long-range distance restraints determined using thisparamagnetic enhancement methodology were sufficient to definethe overall topology of barnase.

Results

Spin labeling and assignment of barnase(H102C)and barnase(H102A0Q15C)

Nitroxide spin labels can be specifically attached to proteins throughdisulfide bond formation with side chains of cysteine residues inthe protein of interest~Berliner et al., 1982; Kosen et al., 1986;Todd et al., 1989!. The fact that wild-type barnase has no cysteineresidues allowed us to introduce a unique cysteine residue at twodifferent solvent accessible positions of barnase, H102C and Q15C,by means of site-directed mutagenesis. It had previously beendemonstrated that mutation of His102 had no dramatic effect onthe stability of the protein~Paddon & Hartley, 1987!. In addition,nonconservative amino acid substitutions over much of the surfaceof barnase were found not to interfere with its binding by barstar,the natural intracellular inhibitor of barnase~Hartley, 1993!. Mod-ification of cysteine residues with MTSSL, shown in Figure 1,generates the spin labeled side chain, generally referred to as R1~Mchaourab et al., 1996!. Assignments for all barnase proteinswere confirmed by NOESY-HSQC experiments~data not shown!.Since amide proton chemical shifts are a sensitive probe of proteinconformation,1H–15N correlation spectra of the monocysteine de-rivatives of barnase were compared with that of the wild-typeprotein~Bycroft et al., 1991!. Figure 2 shows amide proton chem-ical shift differences between wild-type barnase~Jones et al., 1993!and either reduced barnase~H102R1! or reduced barnase~H102A0Q15R1!. No significant amide proton or nitrogen~data not shown!chemical shift differences were observed when compared towild-type barnase suggesting that site-specific introduction of aCys residue and covalent attachment of a nitroxide spin label doesnot significantly affect protein structure. The largest amide protonchemical shift deviation observed was at the site of Cys and MTSSLattachment in barnase~H102C! and barnase~H102A0Q15R1!~Fig. 2!.

The EPR spectra of H102R1 and H102A0Q15R1 in NMR bufferand in NMR buffer with 30% sucrose are shown in Figure 3. In

Fig. 1. Reaction of MTSSL spin label with –SH groups of a protein toproduce the R1 side chain.

Protein global fold determination 303

buffer, the EPR spectra reflect extensive motional averaging of theanisotropic magnetic parameters of the nitroxide due to the rota-tional diffusion of barnase and any internal motions of the nitrox-ide side chain relative to the protein~Fig. 3A,C!. Simulations ofthe spectra in Figures 3A and 3C indicate effective correlationtimes of;1.8 ns at both sites. In the higher viscosity of the sucrosesolution~3 centipoise!, the contribution due to rotational diffusionof barnase is reduced, and the spectral lineshapes are dominated byinternal motions of the side chain~Mchaourab et al., 1996!. Asseen in Figure 3B, the spectrum in sucrose shows a relativelyrestricted motion for H102R1. This degree of restriction is unusualfor a site in a turn and suggests tertiary interactions of the nitroxidewith the protein. For H102A0Q15R1, the spectrum is consistentwith a helix surface site~Fig. 3D! ~Mchaourab et al., 1996!.

Paramagnetic effects

In MTSSL-labeled barnase, resonances belonging to amide pro-tons within 10 Å from the paramagnetic center were usually broad-ened beyond detection at reasonable S0N levels. After the reductionof spin labeled barnase with ascorbate these resonances were eas-ily observed in the1H–15N HSQC spectra. This differential broad-ening is apparent for@15N# barnase~H102A0Q15R1! ~Fig. 4!. Inthe oxidized sample, cross peaks belonging to V10, C15, T16,Y17, D93, W94, and R110 are broadened beyond detection~Fig. 4A!. However, these resonances can be easily found in theascorbate reduced diamagnetic sample~Fig. 4B!.

The longitudinal relaxation times for amide protons in the spin-labeled and corresponding reduced samples were measured in aseries of inversion recovery NMR experiments at 600 and 800 MHz.Peak volumes at individual recovery times were fitted to the three-parameter equation~Ferretti & Weiss, 1989! that accounts for in-complete magnetization inversion and loss of signal during delaysin the conventional HSQC pulse sequence through relaxation ofthe antiphase state Hx,yNz. The paramagnetic contribution from thespin label to the longitudinal relaxation rates~10T1p! was deter-mined by subtraction of the 10T1 for the diamagnetic sample fromthe 10T1 for the paramagnetic sample.

Accuracy of theT1 measurements was assessed from averagingT1s from two different mutants in the reduced forms, which shouldbe similar to measuringT1s on the same protein~Gillespie &Shortle, 1997!, and using the average values for standard deviationcalculations. The standard deviation inT1s for all the data sets at800 MHz was60.01 s. This gave an average error of 10% in10T1p measurements. From the measured values of 10T1p, thecorrelation times for the electron-amide proton vectors were cal-culated from the frequency dependence of paramagnetic effects~Weber et al., 1991! using the Solomon–Bloembergen approxima-tion ~Equation 2!. Values obtained fortc ranged from 0.6 to 1.8 ns.The electron relaxation time, overall tumbling time, correlation timedescribing chemical exchange, and the reorientation time of pro-tons can contribute to the measuredtc parameters. Individualtc val-ues were used for distance calculations. In several cases, due to errorin T1 measurements, the calculation of individual correlation timeswas not possible. In these cases, an average correlation time was used.

It has been suggested thatT1 data interpretation for paramag-netic proteins is complicated because of cross-relaxation processes~Russu & Ho, 1982!. However, in our case, cross-relaxation didnot cause significant averaging of proton longitudinal relaxationtimes as evidenced by comparison ofT1 values for two protonsattached to the same nitrogen. For example, longitudinal relaxationtimes for delta protons belonging to N23 of barnase~H102A0Q15R1! were 1.62 and 1.12 s at 800 MHz. These two protons are

A

B

Fig. 2. Plots of the absolute values of the amide proton chemical shiftdifferences vs. residue number between wild-type barnase and either re-duced barnase~H102R1! or reduced barnase~H102A0Q15R1!. A: Amideproton chemical shift differences vs. residue number between wild-typebarnase and barnase~H102R1!. B: Amide proton chemical shift differencesvs. residue number between wild-type barnase and reduced barnase~H102A0Q15R1!.

A C

B D

Fig. 3. EPR spectra of MTSSL labeled barnase proteins in the presenceand absence of sucrose.A: Barnase~H102R1! in NMR buffer.B: Barnase~H102R1! in NMR buffer containing 30% sucrose.C: Barnase~H102A0Q15R1! in NMR buffer. D: Barnase~H102A0Q15R1! in NMR buffercontaining 30% sucrose. Dashed traces inA andC are spectral simulationsindicating effective correlation times of;1.8 ns in both cases.

304 V. Gaponenko et al.

very close in space and the cross-relaxation effects should be ex-pected to be very strong. This is in agreement with the resultsobtained by Bertini et al.~1997!, who showed that the cross-relaxation averaging of paramagnetic relaxation rates is often tol-erably small. Instances, in which cross-relaxation does become aproblem, could be largely eliminated by perdeuteration of the car-bon bound protons.

Structure calculations

Measured 10T1p values were used in Equation 4 to calculate elec-tron to amide proton distances. Errors in 10T1 measurements werepropagated through the distance calculations resulting in averageerrors for calculated distances of 11%. Experimentally calculateddistances were first compared to those measured from the crystalstructure of barnase~Mauguen et al., 1982! modified to contain the

covalently attached spin label~Fig. 5!. The position of the spinlabel was minimized using 1,000 steps of conjugate gradient min-imization. Differences between the calculated and measured dis-tances on average were 10%. Distances longer than 25 Å werefound to have errors up to 28%.

For both barnase~H102R1! ~Fig. 5A! and ~H102A0Q15R1!~Fig. 5B!, experimentally calculated distances were found to cor-relate with distances measured from the crystal structure of bar-nase. As a consequence of the location of R1 in barnase~H102A0Q15R1!, distances up to 35 Å could be accurately determined fromthis spin label~Fig. 5A!. The correlation between theoretical andexperimental electron to amide proton distances up to 35 Å andthose obtained from the crystal structure of barnase~Fig. 5! dem-onstrates the utility of the paramagnetic probe methodology.

Fig. 4. A: 1H–15N HSQC spectra of oxidized@15N#barnase~H102A0Q15R1!. B: Ascorbate labeled@15N#barnase~H102A0Q15R1!. Sample was;1 mM in protein containing 50 mM deuterated acetate buffer, pH5 4.5and 10% deuterium dioxide. The nitroxide radical was reduced to thehydroxyamine with five equivalents of ascorbate. Spectra were obtained at800 MHz and 308C as described under Materials and methods. Boxes inAcorrespond to amide proton—amide nitrogen cross peaks assigned to res-idues whose amide proton longitudinal relaxation rates are strongly af-fected by the spin label. Boxes inB show the cross peaks that appear in1H–15N correlation spectra upon ascorbate reduction of the spin label.

A

B

Fig. 5. Correlation between theoretical and experimental electron to amideproton distances for(A) barnase~H102R1! and for(B) barnase~H102A0Q15R1!. Theoretical distances were measured from the minimized barnasecrystal structure~Mauguen et al., 1982! containing the attached MTTSLspin label. Experimental distances were determined from paramagneticeffects on the longitudinal relaxation rates andtc values using Equation 4.Errors in experimentally determined distances were estimated byT1 errorpropagation.


A total of 62 experimentally determined distances from barnase~H102R1! and 71 distances from barnase~H102A0Q15R1! out ofpossible 107 distances were used as restraints in calculations. Afull set of distances could not be obtained either due to peakoverlap in1H–15N correlation spectra or large fitting errors inT1calculations.

Using the long-range restraints and the known secondary struc-ture, ensembles of global fold structures wase calculated in X-PLOR.A family of minimized structures was calculated having goodagreement between calculated and experimental electron to amideproton distances. The DALI algorithm permitted the correct bar-nase fold to be distinguished from its mirror image~Holm &Sander, 1993!. Since no side-chain restraints were introduced dur-ing structure calculations, the RMSD for all the backbone atomsshould be appropriate for assessment of the quality of the calcu-lated structures. The RMSD for the average structure calculatedusing the long-range distance and secondary structure restraintsfrom the crystal structure of barnase was 2.9 Å~Fig. 6!. In addi-tion, the global fold structure was able to predict the electrostaticpotential of active site reasonably well. The RMSD using all back-bone atoms for the ensemble of barnase global fold structures was2.2 Å ~Fig. 7!. As can be seen in Figure 7, the loop regions are lessdefined than the regions comprising helices or sheets.

The lowest energy global fold structures had no paramagneticdistance violations greater than 0.5 Å. In addition, 60% of theresidues was within the most favored regions of the Ramachanan-dran map. The same analysis on the high resolution NMR structure~Bycroft et al., 1991! and crystal structure of barnase~Mauguenet al., 1982! found 66 and 82% residues within the most favoredregions of the Ramachanandran map, respectively.

To validate the significance of the long-range distance restraints,an ensemble of five structures was calculated using only secondary

structure restraints. The backbone RMSD between the averagestructure and the crystal structure of barnase was 14.5 Å. TheRMSD between the structures in the ensemble was 12.1 Å. Thus,the experimentally measured long-range restraints are necessaryfor obtaining a well-defined protein fold. In addition, structurecalculations with no secondary structure restraints were also un-dertaken. The backbone RMSD from the crystal structure of bar-nase in this case was 6.5 Å. Thus, the overall topology of barnasecould be determined using only long-range distance restraints. How-ever, the structures calculated with no secondary structure re-straints were generally more extended than the crystal structure ofbarnase. It is worth noting that even though the shape of themolecule was preserved in these calculations, long-range distancerestraints were unable to define the secondary structure elementson their own.a-Helices did not form at all, and some distortionswere introduced into the five-strandedb-sheet.

An alternative approach to estimate thetc values is to utilize thecorrelation time determined by EPR~Fig. 3! since in many casesthe rate with which nuclei sense the magnetic field created by theunpaired electron is dominated by the electronic relaxation rate~Mildvan et al., 1980!. As a test of this approximation, the un-paired electron-amide proton distances were estimated using theuniform correlation time of 1.8 ns, determined from simulations ofthe EPR spectra. The average error on these distances was 13.9%with the largest error amounting to 47.5%. Distances calculated inthis manner have a larger uncertainty than those for distancescalculated using individual correlation times determined from thefrequency dependence of 10T1p. However, a linear correlationbetween the theoretically measured and calculated distances wasstill observed~data not shown!. Moreover, global fold structures ofbarnase could be calculated using these distances as restraints withan average backbone RMSD from the crystal structure of 4.4 Å.

Fig. 6. The average global fold structure of barnase~red! determined using paramagnetic distance restraints overlaid on the crystalstructure of barnase~blue! ~Mauguen et al., 1982!. The RMSD between the two structures is 2.9 Å. Regions shown in red, white, andblue representa-helices,b-strands, and coil structures, respectively.


This observation implies thattc values may not have to be ob-tained using the frequency dependence of the paramagnetic effectsfor calculation of electron to amide proton distances~Equation 3!sufficient for global fold determination. Instead,tc can be simplyestimated using EPR techniques and be utilized for distancecalculations.

Discussion

We have demonstrated that long-range distances, determined fromsite-directed spin labels to amide protons, are sufficient to deter-mine the global fold topology of barnase. Distances up to 35 Å canbe accurately measured~Fig. 4!. Substitution of the R1 side chainat specific solvent accessible sites on barnase was found not toperturb the overall tertiary structure of the protein as monitored bybackbone amide proton and nitrogen chemical shifts~Fig. 2!. EPRspectra of both barnase~H102R1! and ~H102A0Q15R1! showedthat the spin label was mobile and tumbled with atr value of;1.8 ns~Fig. 3!. This value is in the range oftc values calculatedusing the frequency dependence of paramagnetic effects on indi-vidual amide protons.

Linear correlations between theoretical and experimental elec-tron to amide proton distances for both R1 derivatives of barnasewere observed~Fig. 4!. The ease in which long-range distancerestraints can be measured makes this approach powerful for rapidglobal fold determination of proteins and as a supplement to NOE-based distance restraints in the determination of large molecularweight complexes.

Our results demonstrate that it is possible to calculate proteinglobal fold structures using as few as two paramagnetic probes.One possible complication of using only two reference points isthe problem of mirror image structures. However, it was possibleto select the correct folds using three-dimensional structure align-ments looking for known structural characteristics~Holm & Sander,

1993!. In addition, this problem can easily be circumvented ifmore than two paramagnetic probes are used as reference pointsfor protein structure definition. For global fold determination ofproteins with unique structures, greater than two paramagneticprobes may be required. Alternatively, when long-range restraintsderived from paramagnetic relaxation enhancement data are com-bined with a threading algorithm, only a single reference point maybe sufficient to determine the nature of the protein fold.

The use of secondary structure restraints in protein global foldcalculations is important for accurate structure determination. How-ever, the necessity of these restraints should not limit the use of theparamagnetic enhancement methodology. Secondary structure re-straints can easily be obtained from rapid analysis of NOESY datasets. Often NOESY spectra have to be collected to aid the assign-ment process. In addition, the chemical shift index data commonlyused for secondary structure prediction are available as a result ofthe backbone assignment process. Further, as we have shown withbarnase, the overall topology of the protein can be elucidated usingonly long-range distance restraints.

One possible drawback of site-directed spin labeling is the lackof unique sites for introduction of spin labels in proteins withmultiple cysteine residues. However, the techniques for efficientsite-directed spin labeling with minimal perturbations of the back-bone fold, thermal stability, and function are experiencing rapiddevelopment~Hubbell et al., 1998!. We recently were able tosuccessfully spin label human leptin, which naturally contains twostructurally important cysteine residues. Introduction of R1 intohuman leptin was found not to significantly perturb the proteinstructure~E. Abusamhadneh, unpubl. results!.

In conclusion, we have demonstrated the rapid measurement oflong-range paramagnetic distance restraints and the use of second-ary structure restraints are sufficient for determining a proteinglobal fold structure. This is particularly important given the knowl-edge that the factors influencing protein folds are currently poorly

Fig. 7. Ensemble of five global fold barnase structures calculated using paramagnetic distance restraints and the known secondarystructure. The overall backbone RMSD is 2.2 Å.


understood. The lack of this information limits the prospects ofautomated homology modeling. In addition, the use of conven-tional techniques for structure determination by NMR can be laborintensive and inefficient, particularly as the molecular weightincreases.

To circumvent these problems, new approaches that minimizethe time required to determine a global fold and that decrease thedependence on NOE restraints are necessary to increase the effec-tiveness of NMR in global fold determination. The use of para-magnetic probe relaxation methodology in recognition of globalfolds and to provide additional distance restraints in NMR solutionstructure determination is currently underestimated and underuti-lized. The use of site-directed spin and isotope labeling for foldrecognition will become more important as the number of proteinswith unknown function increases.

Materials and methods

Monocysteine barnase proteins

The barnase mutant~H102A! was used in all experiments sincethis mutation inactivates the lethal enzyme and provides higherlevels of protein in the bacterial expression system. Single cysteineresidues were incorporated at either position 15 or 102 of barnaseusing PCR mediated site-directed mutagenesis. The plasmid car-rying the phoA sequence followed by barnase~H102A! was usedto transform BL21~DE3! cells. Barnase~H102C! and barnase~H102A0Q15C! were 15N enriched by growing BL21~DE3! cellswith the appropriate plasmid in minimum media containing15NH4Cl~1 g0L! as a sole source of nitrogen. After induction with IPTG atA6005 0.6, the cells were grown for additional 4 h before harvest-ing. The cell culture was then incubated on ice for 30 min in thepresence of 0.2 M acetate to allow barnase trapped in the periplas-mic space to be released into the media. Cell debris was removedby centrifugation, and the barnase proteins purified from the su-pernatant by absorption on SP-Trisacryl beads. Proteins were elutedwith 1 M NaCl and then dialyzed against distilled water overnight.The barnase proteins were concentrated and found to be homo-geneous by SDS-polyacrylamide gel electrophoresis and stainingwith Coomassie Brilliant Blue. Purified monocysteine proteins werealso analyzed by gel electrophoresis under nondenaturing condi-tions to monitor any possible disulfide linked dimer formation.Barnase~H102C! or barnase~H102A0Q15C! did not exhibit di-sulfide linked dimer formation and incubation at room temperatureovernight in the presence of 10 mM DTT prior to spin labeling wasnot necessary. Nitroxide spin labeling was initiated by adding asevenfold molar excess of MTSSL dissolved in 40mL of acetoneand incubated at room temperature overnight. Excess spin labelwas removed from the protein by gel filtration on a P4 column andthe protein lyophilized. The spin labeled protein was dissolved toprovide an;1 mM solution in NMR buffer containing 50 mMdeuterated acetate buffer, pH5 4.5 and 10% deuterium oxide. Theefficiency of spin labeling was determined by the means of1H–15N HSQC correlation experiments and double integration of theEPR spectrum. In all cases, proteins were found to be.90% spinlabeled. To reduce the paramagnetic nitroxide spin label, a three-fold molar excess of ascorbate was added and the sample incu-bated at room temperature overnight. The pH was carefullyreadjusted to 4.5 before collecting NMR data on the reduced spinlabeled proteins.

EPR measurements and simulations

EPR spectroscopy was performed on a Varian E-109 spectrometerfitted with a two-loop one-gap resonator~Hubbell et al., 1987!. A5 mL sample was loaded into a 0.84 mm o.d. capillary that wassealed on one end. Spectra were acquired using a 2 mWincidentmicrowave power. The effective correlation time was estimated byspectral simulations~Budil et al., 1996!.

NMR spectroscopy

All NMR spectra were collected at 308C on Varian 500, 600, and800 NMR spectrometers. Resonance assignments for wild-typebarnase were taken from Jones et al.~1993!. Assignments for@15N#barnase~H102A!, ascorbate reduced barnase~H102R1!, and ascor-bate reduced barnase~H102A0Q15C! were confirmed on the15N-enriched proteins by NOESY-HSQC experiments at 800 MHz witha mixing time of 70 ms.T1 inversion recovery1H–15N HSQCspectra were collected with delay times of 10, 200, 400, 600, 800,1,000, 1,400, 1,800, 2,200, and 3,000 ms. Three double pointswere also collected at 200, 1,400, and 2,200 ms to estimate theerror inT1 measurements. Spectra were processed using the Felixsoftware~MSI, San Diego, California! with resolution enhance-ment ~908 shifted sine-bell squared function in both dimensions!.Peak volumes were measured for both the spin-labeled and ascor-bate reduced forms of the barnase proteins. The values were fittedto the following equation~Ferretti & Weiss, 1989!:

V~t! 5 VD @12 B~12 exp~2k0T1! ! 3 exp~2t0T1! # ~1!

wherek is the sum of acquisition and preparation times,B is anadjustment parameter for incomplete magnetization inversion,t isthe recovery delay, andT1 is longitudinal relaxation time.

Paramagnetic effects on amide proton relaxation rates were cal-culated using

1

T1p

51

T1para

21

T1dia

~2!

whereT1p is the paramagnetic effect on the longitudinal relaxationtimes, T1para is the longitudinal relaxation time of the oxidizedform of the spin labeled protein, andT1dia is the longitudinal timeof the ascorbate reduced spin labeled protein~Mildvan et al., 1980!.

Individual correlation timestc for the amide protons were esti-mated from the frequency dependence of the paramagnetic effectsat 600 and 800 MHz. The following equation was used to calculatetc ~Weber et al., 1991!:

tc2 5

T1p8002 T1p600

T1p600v6002 2 T1p600v800

2. ~3!

Estimates in the distancer are relatively insensitive to small errorsin tc, sincer depends on the sixth root of the correlation time.

Distances were calculated using the following form of theSolomon–Bloembergen equation~Solomon, 1955!:

r 6 52K

T1p

33tc

11 v2tc2

~4!

whereK is a constant, 1.233 1032 cm6 s22.


The parameter and topology files describing structural charac-teristics of the MTSSL spin label were obtained from the UppsalaUniversity, Sweden. The global fold structure of barnase was cal-culated using distance geometry0simulated annealing protocols inX-PLOR-3.1~Brünger, 1992!. Distances between the unpaired elec-tron in the R1 side chain and the amide protons of barnase weregiven the energy function normally used for NOE restraints inX-PLOR. This is possible since both NOE and paramagnetic distancerestraints have anr 26 distance dependence. Lower and upper boundswere derived by propagation of errors inT1 measurements to thecalculated unpaired electron-amide proton distances. All other pa-rameters in the distance geometry0simulated annealing protocolswere essentially those of Nilges et al.~1988a, 1988b!. Structureswere calculated from starting templates with a fully extended ge-ometry. Embedded substructures were optimized using simulatedannealing and refined using 6,000 rounds of restrained minimiza-tion. The quality of the structures generated were evaluated by theprogram PROCHECK~Laskowski et al., 1993!.

Acknowledgments

This work supported by Grants HL 41496 and AR44324~to P.R.R.! and EY05216 ~to W.L.H.! from the National Institutes of Health and USPHSNational Research Service Award GM 08496.

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