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Unformatted text preview: Proc.Natl.Acad. Sci. USA Vol.90,pp. 3275-3279, April1993 Biophysics The a aneurism: A structuralmotifrevealedin an insertionmutant of staphylococcal nuclease LISA J. KEEFE*t, JOHN SONDEKt§,DAVID SHORTLEt, AND EATON E. LATTMAN*¶ Departmentsof*Biophysicsand BiophysicalChemistryand*BiologicalChemistry,TheJohnsHopkinsUniversitySchoolofMedicine,Baltimore, MD 21205-2185 Communicated byThomas J.Kelly,December 16,1992 ABSTRACT The x-ray crystal structure ofa mutant of staphylococcalnucleasethatcontainsasingleglycineresidue insertedintheC-terminal a-helixhasbeen solvedto1.67A resolutionand refinedtoa crystallographic R valueof0.170. Thisinsertedglycineresidueisaccommodatedinthea-helixby formation ofa previously uncharacterized bulge, which we term the a aneurism. A conformational search of known proteinstructureshasidentifiedtheaaneurisminanumberof proteinfamilies,includingthehistocompatibilityantigensand hemoglobins. Theenzyme nucleasefromStaphylococcusaureusisapow- erful system inwhich mutagenesis can be used to probe protein stability and folding (1-6). It comprises a single polypeptidechainof149aminoacidresidueswithno disul- fidelinks.Recently,solutionstudiesof a seriesofinsertion mutations introduced into the enzyme (7-9) have raised severalimportantstructuralissues.Insertionmutationsare differentfromsubstitutionmutationsinthat,outofnecessity, the backbone of the protein must be perturbed. It was thereforeanticipatedthatinsertionmutationswouldbequite destabilizing. In fact,from a study ofchanges in stability produced by more than 50 different insertion mutations, staphylococcal nuclease appears tobe highlyadaptablein accommodating insertions of amino acid residues into a varietyofsecondarystructuralelements(7,8).On average, theeffecton stabilityoftheinsertionofasinglealanineor glycine residue issimilarto the effect on stabilityofthe substitution foralanine or glycine ofone ofthe residues flankingtheinsertionsite(7).Moreover, mutants inwhich twoglycineresidues,oronealanineresidueandoneglycine residue,have been insertedalmostalways have stabilities similartothoseofsingleinsertionsatthesame site(8).The oneexceptiontothisistheseriesof insertionmutationsata siteinthesecondturnoftheC-terminala-helixofnuclease, betweenwild-typeresiduesArg-126andLys-127.Here,the two-residue insertion mutants are significantlyless stable thanthesingle-residueinsertionmutants(8).Intheabsence ofstructural information, itisdifficultto predicthow an insertion in an a-helix isaccommodated. A register shift within the helix to include the insertion would alter the patternofhydrophilicandhydrophobicside-chainpositions andrearrangecontactsbetweenhelixsidechainsandtherest ofthe proteinmolecule. Alternatively, a disruptionofthe helix would cause loss of hydrogen bonds and possibly formation ofunfavorable conformations. To elucidate the structural perturbations that must accompany these inser- tions,thecrystalstructureofthemutant 126G127, inwhich a singleglycineresidue isinserted,has been determined.llsingleglycineresidue isinserted,has been determined....
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