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m .«? v«v v^ ^V s^/ x*V x ; X \ ,; ; ^ v ^ #v % ^v %\\^fp\^ .«. .... .vV ti. -—& ^ ^ #>^% ^ * a«=a« ^ ^ £t>e<'sl ^ ^ «£—?* ^ ^ f^% ^ *v«^£-"£ ^ y ^ V v y _ v y _ v *««$* vr „m. s^ .# NATIONAL INSTITUTES OF HEALTH ~ >' N—IH LIBRARY H. 6 tS ^c->- 1995 X ^ <k ,** > 1/ BLDG10, 10CENTER OR. BETHESDA.MD 20892-1150 ^V ^ Am x x x s x ^ ^ ^V Asf^ ^«*^- ** > X / \ / \ / X / \ »% / ,^& *»% *>> CN3*. ^ 4ft. «E>^ x x • x x x * +* /Rv **' /a\ /a </' /a* ,/>» / a \*r«£6\>r<!a\^<\^^*»V'"*% ^/\ ft, % r rjs • / x'f(^&)S s X 1 X^/X''^ / A*»^ « t vx «v*w j> x^il^w.# s v ^W /-* v* ^\W^J • x ^«* s- xyC&Kf s \ y ^ ,07\ w ^ \ • \ y V \ • ^-^ v**^y x > Vj ./ «E*i> j>^.«. \, .y ..... .«. y *^ # ^ #v X / \ ^" • \ ^* •>> \ <"e^ • \ H • :\'^<^! •>> <? % ^ ^ \ % **** \W, ^ '^ X ^ x x y jrr/Rv ^ ^ «\ ^ «.# n\ *4f «*Rv ' MblETt MUM1EI ptr<um«t»fforiaAiniAWKiMAMKtv»ct>-n»ucHrjaTMittv>ci NOTICE OF INTRAMURAL RESEARCH PROJECT Z01 DK 11000-01 ODIIv KXtOS COVBXD October 1, 1993 through September 30, 1994 TmxOF PROJECT »jgaSU - to aLMeJunjBUu »"P — »—'" I Physics of Ionic Channels and other Proteins with Aqueous Cavities P.I.: V.A. Parsegian Guest Researcher Others: S.M. Bezrukov Visiting Scientist LBM, NIDDK J.J. Kasianowicz Guest Researcher LBM, NIDDK D.C. Rau Research Chemist LBM, NIDDK R. Brutyan Special Volunteer LBM, NIDDK COOPOUTKC Wtn flfTl Office of Naval Research, Arlington, VA (Dr. I. Vodyanoy); University of Maryland, College Park, MD (Dr. M. Colombini); Monell Chemical Senses CenUr, Philadelphia, PA (Dr. A.M. Feigin) , tAMKANCN Division of Intramural Research SCTX>S Biophysics Unit *nmrrE andlocation NIDDK, NIH, Bethesda, Maryland 20892 totaln*rr «am ftOFEUIONAL OTHEIt: 4.0 4.0 O0£V A»*OP*.IATt »OXIU> D(a) Human subjects D(b) Human tissues D(c) Neither D(al) Minors Of22) Tnterviews w tUMMAjtY OF W0R>. (V» wd.* gjgjjd gg t>- ww n»/ Ut >i HJ i Channels made from the peptide alamethicin have been observed while subjected to the osmotic action of differently sized neutral polymers. It is possible not only to see the degree of penetration of the polymers into the channel from their osmotic action but also to follow the- kinetics of motion of small polymers through the ionic channel. These channels are sensitive to the phospholipid of the bilayer into which they are incorporated, in particular there is a strong correlation between the probability of high-conductance states and the tendency of the phospholipid to form non-lamellar structures. Hofmeister effect is shown to apply to transport properties of ionic channels. Chaotropic anions bind to roflamycoin channels for longer times, increase their conductance and induce cationic selectivity according to their position in Hofmeister series. In experiments on hemoglobin oxygenation, it is demonstrated that for weak binding reactions the osmotic contributions to binding can be as significant as the direct contribution from solute binding. rm tocA» M2> r»ojtrt MUMttt KMKTMtwrorMi<aTiiMCinriMitivKttntiKWjaniiunct' NOTICE OF INTRAMURAL RESEARCH PROJECT |Z01 DK 11001-01 ODIR fOUOS COvXXCD October 1, 1993 to September 30, 1994 TJTUOF PtOJETT IK »i«» te. Trtw. fr w m, to, fa^ ». fcjfcjl Structure and Physical Properties of DNA and DNA-Protein Complexes mPs.cIv.A:i pvoDt.kCa.toiRa<lu-- «)» Bfeaipi »w.i fete *» »—ReIsteaamracahi *C"heMmuisMtaaa < fc im iTiii0rDi, NIDDK Others: N.Yu. Sidorova Guest Researcher 0D, NIDDK cookxatwc iwmjiLBJ LPTB, NICHD (Dr. M. Garner); Towscn State University, Towson, MD (Dr. R. Preisler); University of Nevada, Reno, NV (Dr. R. Harrington): LCB, NHLBI (Dr. E. Korn> LMMANCM Division of Intramural Research nmo* Section on Molecular Biophysics ISSTTTtTt AKDLOCATION NIDDK, NIH, Bethesda, Maryland 20892 TOTALrTAFT rtAJti ftOfEUIOkAL: CTNU: 1.5 1.5 CHECK AWOMUAU tOXIUi D(a) Human subjects D(b) Human tissues D(c) Neither D(al) Minors Ofa?) Interviews Direct measurement of forces between mplecules in condensed arrays show a dominating contribution from water structuring. Now measurements of the offmlic sensitivity of DNA-protein and DNA-drug complex formation in dilute solution are also showing a link between binding strength and structured vater •release." Binding constants for the specific.association of galar-trap repressor with operator and far nonspecific binding differ by a factor of 10 . This difference is reflected in the release of some 150 water molecules for operator binding compared with nonspecific association. Even much snaller binding energy differences of repressor to different operator sequences are seen as differences in water release. The binding constant of the gal repressor-CL operator association is twice that of the repressor-Oj ccnplex and is accompanied by the relative release of 6 water ndecules. A connection between energy and water release is also seen for drug-£KA interactions. Ihe binding constant of netropsin to an Eco RI site is 10 times stronger than to an Nde I site end also releases sue 30 more water molecules. An important role for a change in hydration accompanying a nucleic acid conformational transition has been uncovered. Ihe sensitivity of the B-Z transition to water activity indicates that a difference in the number of solute excluding water molecules between the two conformations is central to the transition. Conditions in the cell ray be sufficiently crowded to allow the Z-form to exist without other stresses. Changes in the internal flexibility of Acanthamoeba myosin II minifilaments with AIP binding have been further characterized. Minifilaments with bound AIP are about 50 fold more flexible than with either no bound nucleotide or bound ADP. Changes in flexibility are apparently concomitant with the force generating step. Bending relaxation times extracted the electric birefringence curves indicate that flexing is occurring at the Sl-52 junction. Coupled with our previous experiments, a previously unsuspected tight coupling of the HtHMi and the S1-S2 junction conformations probably exists. PHI DCIIn.M]! PROJECT NUMBER DEPARTMENT OFHEALTH ANDHUMAN CERVICES -PUBLIC HEAL. H SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZD1 IK 11500-01 CDIR PERIOD COVERED October 1, 1993 through September 30, 1994 mi TITLE OF PROJECT (80cftonctft ortot. TnMmuttfhononetnetlli tmborders-) DNA Repair and Replication in Xenopus MaW PRINCIPAL MVESDGATOR CJrrotttorprotoatonm porwommbttowthtPrindp*ImmtxigmtofJ Petmt, Mfc, iy, andJvt/o/t*mtmnlonl PI: Eric Ackerman Special Expert OD, NXDDK Others: Haoko Oda Visiting Fellow OD, KZ7JDK Alexander Spoonde Visiting Fellow OD, HXDDK Jing Zhang IRTA OD, HXSDK COOPERATING ft«TB »any) LAB/BRANCH Office of Director SECTION INSTITUTE ANDLOCATION NIH, NIDDK, Bethesda, MP 20B92 TOTALSTAFF YEARS: PROFESSIONAL: OTHER: 2.0 2.0 CHECK APPROPRIATE BOXtES) O (a) Human subjects D (b) Human tissues D (c) Neither D (a1) Minors D fa2) Interviews SUMMARY OFWORK (Utttfndardumducad typo. Donotmxeood the tpoctprovided.) During the extensive laboratory renovations following our move to Bldg. 5, there was a complete change in laboratory personnel. For most of the year, only one visiting fellow was present. These events temporarily slowed our progress towards understanding the molecular biology of vertebrate DNA repair. During early development Xenopus replicates its DNA nearly as fast as E. coli in log phase. We demonstrated that oocytes are an excellent source of both DNA repair activity and single-strand to double-strand replication activity. Pyrimidine dimer and (6,4) photoproduct repair was shown by microinjecting UV-irradiated plasmid DNA into oocytes or adding damaged DNA to an extremely efficient extract derived from oocytes. We have also studied DNA repair for alkylated and chemically modified DNA as well as DNA replication with our repair extract. One of the advantages of the Xenopus system is that we can study repair/replication reactions both in living cells by microinjection and in extremely efficient extracts. Unlike other extracts that repair <2% of the input damaged DNA, our extracts repair all of the damaged substrate.