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Protein global fold determination using site-directed spin and

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  • Protein global fold determination using site-directedspin and isotope labeling



    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!


    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 amide 1H 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 werecalculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1Hcorrelation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structurecalculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints,barnase global folds were calculated having backbone RMSDs ,3 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!, 9:302309. Cambridge University Press. Printed in the USA.Copyright 2000 The Protein Society


  • protons, which are restricted to distances under 6 . Nitroxide spinlabels can provide long-range distance restraints up to 35 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 the r 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 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 on D-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 estimated using the SolomonBloembergenequations ~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 by Bacilus 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 SolomonBloembergen 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 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.


    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 cystein

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