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• W1583012020 abstract "α1-Antitrypsin is the most abundant circulating protease inhibitor and the archetype of the serine protease inhibitor or serpin superfamily. Members of this family may be inactivated by point mutations that favor transition to a polymeric conformation. This polymeric conformation underlies diseases as diverse as α1-antitrypsin deficiency-related cirrhosis, thrombosis, angio-edema, and dementia. The precise structural linkage within a polymer has been the subject of much debate with evidence for reactive loop insertion into β-sheet A or C or as strand 7A. We have used site directed cysteine mutants and fluorescence resonance energy transfer (FRET) to measure a number of distances between monomeric units in polymeric α1-antitrypsin. We have then used a combinatorial approach to compare distances determined from FRET with distances obtained from 2.9 × 106 different possible orientations of the α1-antitrypsin polymer. The closest matches between experimental FRET measurements and theoretical structures show conclusively that polymers of α1-antitrypsin form by insertion of the reactive loop into β-sheet A. α1-Antitrypsin is the most abundant circulating protease inhibitor and the archetype of the serine protease inhibitor or serpin superfamily. Members of this family may be inactivated by point mutations that favor transition to a polymeric conformation. This polymeric conformation underlies diseases as diverse as α1-antitrypsin deficiency-related cirrhosis, thrombosis, angio-edema, and dementia. The precise structural linkage within a polymer has been the subject of much debate with evidence for reactive loop insertion into β-sheet A or C or as strand 7A. We have used site directed cysteine mutants and fluorescence resonance energy transfer (FRET) to measure a number of distances between monomeric units in polymeric α1-antitrypsin. We have then used a combinatorial approach to compare distances determined from FRET with distances obtained from 2.9 × 106 different possible orientations of the α1-antitrypsin polymer. The closest matches between experimental FRET measurements and theoretical structures show conclusively that polymers of α1-antitrypsin form by insertion of the reactive loop into β-sheet A. 5-iodoacetamidofluorescein tetramethylrhodamine-5-iodoacetamide fluorescence resonance energy transfer reactive center loop α1-Antitrypsin is synthesized in the liver and secreted into the plasma where it is the most abundant circulating protease inhibitor. It is the archetypal member of theserine protease inhibitor or serpin superfamily (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (828) Google Scholar), and like other members of this family it shares a common molecular structure based on a mobile reactive center loop and a five-stranded β-sheet A (2Elliott P.R. Lomas D.A. Carrell R.W. Abrahams J.-P. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (239) Google Scholar, 3Schreuder H.A. de Boer B. Dijkema R. Mulders J. Theunissen H.J.M. Grootenhuis P.D.J. Hol W.G.J. Nat. Struct. Biol. 1994; 1: 48-54Crossref PubMed Scopus (267) Google Scholar, 4Carrell R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 5Sharp A.M. Stein P.E. Pannu N.S. Carrell R.W. Berkenpas M.B. Ginsburg D. Lawrence D.A. Read R.J. Structure. 1999; 7: 111-118Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The reactive loop acts as a peptide “bait” for the cognate protease, and after docking the loop is cleaved and the acyl intermediate is inserted into β-sheet A. This major conformational change results in the translocation of the protease to the end of the molecule distal to the initial docking site (6Stratikos E. Gettins P.G.W. J. Biol. Chem. 1998; 273: 15582-15589Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Wright H.T. Scarsdale J.N. Proteins. 1995; 22: 210-225Crossref PubMed Scopus (156) Google Scholar, 8Wilczynska M. Fa M. Karolin J. Ohlsson P.-L. Johansson L.B.-A. Ny T. Nat. Struct. Biol. 1997; 4: 354-357Crossref PubMed Scopus (137) Google Scholar) and its inactivation by distortion of the catalytic triad (9Plotnick M.I. Mayne L. Schecter N.M. Rubin H. Biochemistry. 1996; 35: 7586-7590Crossref PubMed Scopus (88) Google Scholar). Mobility of the reactive loop is essential for inhibitory function but also favors aberrant conformations associated with disease (10Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (389) Google Scholar). In particular the loop is able to insert into the β-sheet of a second molecule to form well ordered polymers that are the basis of the profound plasma deficiency of the Z (11Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar), Siiyama (12Lomas D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar), and Mmalton (13Lomas D.A. Elliott P.R. Sidhar S.K. Foreman R.C. Finch J.T. Cox D.W. Whisstock J.C. Carrell R.W. J. Biol. Chem. 1995; 270: 16864-16870Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) variants of α1-antitrypsin. The polymerized protein accumulates in the endoplasmic reticulum of the hepatocyte to form inclusions that are associated with juvenile hepatitis, cirrhosis, and hepatoocellular carcinoma (14Eriksson S. Carlson J. Velez R. N. Engl. J. Med. 1986; 314: 736-739Crossref PubMed Scopus (471) Google Scholar). The accompanying plasma deficiency predisposes the Z homozygote to early onset emphysema (15Eriksson S. Acta Med. Scand. 1965; 432 (suppl.): 1-85Google Scholar). Loop sheet polymers have also been reported with dysfunctional mutants of C1-inhibitor (16Aulak K.S. Eldering E. Hack C.E. Lubbers Y.P.T. Harrison R.A. Mast A. Cicardi M. Davis III, A.E. J. Biol. Chem. 1993; 268: 18088-18094Abstract Full Text PDF PubMed Google Scholar), α1-antichymotrypsin (17Gooptu B. Hazes B. Chang W.-S.W. Dafforn T.R. Carrell R.W. Read R.J. Lomas D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 67-72Crossref PubMed Scopus (175) Google Scholar), and antithrombin (18Bruce D. Perry D.J. Borg J.-Y. Carrell R.W. Wardell M.R. J. Clin. Invest. 1994; 94: 2265-2274Crossref PubMed Scopus (147) Google Scholar) in association with angio-edema, emphysema, and thrombosis, respectively, and polymers of neuroserpin underlie a novel inclusion body dementia (19Davis R.L. Shrimpton A.E. Holohan P.D. Bradshaw C. Feiglin D. Collins G.H. Kinter J. Sonderegger P. Becker L.M. Lacbawan F. Krasnewich D. Muenke M. Lawrence D.A. Yerby M.S. Shaw C.M. Gooptu B. Elliott P.R. Finch J.T. Carrell R.W. Lomas D.A. Nature. 1999; 401: 376-379Crossref PubMed Google Scholar). Moreover, this conformational transition occurs spontaneously in plasminogen activator inhibitor-2 and is likely to be important in the control of intracellular proteolysis (20Mikus P. Urano T. Liljeström P. Ny T. Eur. J. Biochem. 1993; 218: 1071-1082Crossref PubMed Scopus (74) Google Scholar). The precise protein-protein linkage that underlies polymer formation remains unclear. Polymerization of Z α1-antitrypsin can be blocked by peptides that are homologous to the reactive center loop by annealing to β-sheet A (11Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar, 21Skinner R. Chang W.-S.W. Jin C. Pei X. Huntington J.A. Abrahams J.-P. Carrell R.W. Lomas D.A. J. Mol. Biol. 1998; 283: 9-14Crossref PubMed Scopus (93) Google Scholar). It was therefore proposed that polymers were formed by the insertion of the loop of one molecule into the β-sheet A of another. The crystal structure of an antithrombin dimer revealed another mechanism with the loop of one molecule inserting to replace strand 1 of β-sheet C of a second molecule (3Schreuder H.A. de Boer B. Dijkema R. Mulders J. Theunissen H.J.M. Grootenhuis P.D.J. Hol W.G.J. Nat. Struct. Biol. 1994; 1: 48-54Crossref PubMed Scopus (267) Google Scholar,4Carrell R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Support for this hypothesis came from the antithrombin variant Rouen VI (18Bruce D. Perry D.J. Borg J.-Y. Carrell R.W. Wardell M.R. J. Clin. Invest. 1994; 94: 2265-2274Crossref PubMed Scopus (147) Google Scholar) and the Mmalton variant of α1-antitrypsin (13Lomas D.A. Elliott P.R. Sidhar S.K. Foreman R.C. Finch J.T. Cox D.W. Whisstock J.C. Carrell R.W. J. Biol. Chem. 1995; 270: 16864-16870Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), which were predicted to form short chain polymers terminated by a β-sheet C linkage. Epitope mapping of monoclonal antibodies specific to C1-inhibitor also suggested polymer formation via a C-sheet mechanism (22Patston P.A. Hauert J. Michaud M. Schapira M. FEBS Lett. 1995; 368: 401-404Crossref PubMed Scopus (42) Google Scholar). More recently the crystal structure of plasminogen activator inhibitor-1 has raised the possibility of polymerization in which the reactive loop anneals as strand 7A (5Sharp A.M. Stein P.E. Pannu N.S. Carrell R.W. Berkenpas M.B. Ginsburg D. Lawrence D.A. Read R.J. Structure. 1999; 7: 111-118Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We have used recombinant α1-antitrypsin with three cysteine variants to determine the structural mechanism of polymer formation. The data show conclusively that α1-antitrypsin polymers form by a reactive loop:β-sheet A linkage. Wild type Pittsburgh α1-antitrypsin (M358R) and three cysteine variants (S121C, D159C, and I360C) were prepared and cloned into the pET16b plasmid as detailed previously (6Stratikos E. Gettins P.G.W. J. Biol. Chem. 1998; 273: 15582-15589Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The S121C, D159C, and I360C cysteine variants also carried the C232S mutation, ensuring that all mutants contained only one free cysteine residue. The α1-antitrypsin sequence of each plasmid was confirmed by dideoxynucleotide sequencing. Each plasmid containing the mutated α1-antitrypsin was transformed into BL21(DE3) cells (Novagen). The recombinant proteins were expressed and purified from inclusion bodies as detailed previously (6Stratikos E. Gettins P.G.W. J. Biol. Chem. 1998; 273: 15582-15589Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Purity was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis, and the proteins were dialyzed into 50 mm Tris, 50 mmKCl, 1 mm dithiothreitol, pH 7.4 and stored at −80 °C until required. SDS and nondenaturing polyacrylamide gel electrophoresis, inhibitory activity, and measurement of the rate of polymerization were performed as detailed previously (23Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The proteins were labeled with 5-iodoacetamidofluorescein (5-IAF)1 and tetramethylrhodamine-5-iodoacetamide (5-TMRIA) according to the manufacturer's instructions (Molecular Probes Inc.). The labeled protein was separated from unreacted label with a NAP-10 (Amersham Pharmacia Biotech) gel filtration column. The 5-IAF labeling of each variant was adjusted to 50% by addition of the corresponding unlabeled mutant. All fluorescence measurements were made using a PerkinElmer Life Sciences LS-50B spectrofluorimeter. Spectra were measured at 25 °C using slit widths of 2.5 nm for the excitation beam and 4.0 nm for the emitted beam. The 5-IAF label was excited at 495 nm, and emitted light was measured between 500 and 600 nm. Energy transfer between 5-IAF and 5-TMRIA was measured by recording the decrease in intensity of light emitted from 5-IAF. This method was used instead of measuring changes in acceptor signal, because the signal obtained after subtraction of the appropriate controls was significantly larger. This allowed an increase in the accuracy of the measurement and hence a decrease in the error for later calculations. The efficiency of transfer was calculated using the following relationship. E=1−FD,AFDEquation 1 where F D,A represents the fluorescence of the donor (5-IAF) in the presence of acceptor (5-TMRIA) andFD is the fluorescence of the donor only.F D,A was measured as the emission spectra of a mixture of donor and acceptor labeled protein excited at 490 nm (slit widths were 2.5 nm excitation, 4 nm emission) after incubation at 45 °C for 48 h. The spectrum was normalized for the contribution of non-FRET acceptor emission by subtraction of the spectrum of a polymerized sample containing labeled acceptor and unlabeled donor. F D was obtained by measuring the fluorescence of a solution containing the labeled donor with unlabeled acceptor that had undergone the same incubation. Adjustments were made for the effect of less than 100% of the acceptor being labeled with 5-TMRIA (50% labeling for Cys360 and 20% for Cys159) by adjusting the intensity F D,Ausing the following relationship.FD,A(corrected)=FD,A(observed)−(1−p)FDpEquation 2 where p is the fraction of labeled acceptor. The distance between two fluorophores participating in the fluorescence resonance energy transfer is defined as follows.R=(1/E−1) 1/6R0Equation 3 where R 0 is defined as the distance over which only 50% transfer would occur and E is the measured FRET efficiency. R 0 is defined as follows.R0=((8.79×10−5)κ2n−4φDJDA)6Equation 4 where κ2 is the orientation factor and is given the value of 23 (25Stratikos E. Gettins P.G.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4808-4813Crossref PubMed Scopus (216) Google Scholar), indicating that both donor and acceptor exhibit isotropic motion (24Fairclough R.H. Cantor C.R. Methods Enzymol. 1978; XLVIII: 347-380Crossref Scopus (297) Google Scholar); n is the refractive index term and is routinely given a value of 1.4; JDA is the spectral overlap integral term for the donor acceptor pair and has been determined to be 0.5 m−1cm−1 nm4 for 5-IAF with 5-TMRIA (24Fairclough R.H. Cantor C.R. Methods Enzymol. 1978; XLVIII: 347-380Crossref Scopus (297) Google Scholar); andφD is the quantum yield of the donor which is measured with respect to that of sodium fluorescein (10−6m in 0.01 n NaOH, pH 12,φ = 0.79) (26Taylor D.L. Reidler J. Spudich J.A. Stryer L. J. Cell Biol. 1981; 89: 362-367Crossref PubMed Scopus (96) Google Scholar). The quantum yield was measured for each labeling position (121, 159, 232, and 360) and ranged from 0.59 to 0.23 giving R 0 values from 34 to 40 Å. Quenching experiments were carried out on the four fluorescein labeled proteins before and after polymerization. A 0.5 mg/ml solution of each protein was split into two aliquots; one aliquot was incubated at 45 °C for 48 h, and the other was stored at 4 °C. The change in fluorescence of the fluorescein probe on each protein was then measured (excitation, 485 nm; emission, 530 nm) during the addition of 6 m acrylamide to a final concentration of 1 m. Acrylamide was chosen as the quenching agent because its polar quality restricts quenching to fluorophore moieties on the surface of the protein (27Sherwin, S. S., and Leavis, P. C. Methods Enzymol., XLIX, 222–236.Google Scholar, 28Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1999: 237-265Crossref Google Scholar, 29Eftink M.R. Ghiron C.A. Anal. Biochem. 1981; 114: 199-227Crossref PubMed Scopus (1608) Google Scholar). Unlike the charged quenchers such as iodide, acrylamide shows little sensitivity to the electrostatic environment around the fluorophore. The fluorescence changes were then rescaled to take into account any error because of dilution, and the resulting quenched data were plotted. These data were analyzed with respect to the following Stern-Volmer equation (27Sherwin, S. S., and Leavis, P. C. Methods Enzymol., XLIX, 222–236.Google Scholar). I0/I=1+ksv[Q]Equation 5 where I 0 is the initial fluorescence,I is the fluorescence intensity at concentration Q of quenching agent, and kSV is the Stern-Volmer constant. Our recent 2.0 Å structure of α1-antitrypsin (30Elliott P.R. Pei X.Y. Dafforn T.R. Lomas D.A. Protein Sci. 2000; 9: 1274-1281Crossref PubMed Scopus (169) Google Scholar) was used as the monomeric unit to construct a polymer. All modeling was performed using Insight II 97.0 and CharmM (MSI, San Diego, CA). An initial approximation of the structure of the polymer was obtained by performing an exhaustive search of all possible polymer structures. This was achieved by searching the six spatial degrees (three translational and three rotational) of freedom that define the relative positions of monomers within a polymer using the following method: 1) A cube was defined with 120 Å long sides positioned on the center of mass of an α1-antitrypsin molecule. 2) The cube was split up into a grid along all three axes with a spacing of 10 Å. 3) A second α1-antitrypsin molecule was translated to a point on the grid. 4) The second α1-antitrypsin was rotated about its center exploring all three axes of rotational space at 30° intervals. 5) For each rotation a new grid was defined with the second α1-antitrypsin at its center. 6) A third α1-antitrypsin molecule was placed using the same translation and rotations that were used to place the second molecule. 7) Steps 5 and 6 were repeated until a 7-mer polymer was created. 8) Theoretical transfer efficiencies (E) were calculated between the fourth monomer in the polymer and all other monomers using Equation 3 and the methods of Miki and Iio (31Miki M. Iio T. Biochim. Biophys. Acta. 1984; 790: 201-207Crossref PubMed Scopus (11) Google Scholar). 9) The totalE value was calculated, and the structure was saved if it matched the experimental values. 10) The procedure was repeated allowing exploration of all possible relative orientations at each grid point. Implementation of this method using translational grid spacings of 10 Å and rotational grid spacings of 30° led to 126(2.9 × 106) conformations being searched. For a conformation to be selected as a candidate for the real polymer structure, five of seven of the theoretical E values calculated for a conformation had to be within 0.05 of the experimental value, and the resulting polymer had to be sterically sensible. The reactive center loop between the P3 and P8 residues was modeled into a β-strand to complement insertion into either β-sheet A or C of the N+1 molecule. A- and C-sheet polymers were built according to the structures proposed previously (2Elliott P.R. Lomas D.A. Carrell R.W. Abrahams J.-P. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (239) Google Scholar, 32Chang W.-S.W. Whisstock J. Hopkins P.C.R. Lesk A.M. Carrell R.W. Wardell M.R. Protein Sci. 1997; 6: 89-98Crossref PubMed Scopus (72) Google Scholar). Measurements of solvent accessible surface area were carried out using Getarea 1.1 (33Fraczkiewicz R. Braun W. J. Comp. Chem. 1998; 19: 319-333Crossref Scopus (848) Google Scholar) with a probe radius of 1.4 Å. Wild type Pittsburgh α1-antitrypsin (M358R) and the S121C, D159C, and I360C variants were purified to homogeneity and migrated as a single band on SDS-polyacrylamide gel electrophoresis. They were 45, 71, 70, and 77% active, respectively, as inhibitors when assessed against bovine α-chymotrypsin. The polymerization rates of the cysteine variants of α1-antitrypsin in both native and derivatized form (with covalently bound 5-TMRIA) were measured by monitoring changes in both intrinsic tryptophan fluorescence and fluorescence of the bound probe (Table I) and were confirmed by nondenaturing polyacrylamide gel electrophoresis. The values were similar to those of wild type recombinant α1-antitrypsin (358M), but polymerization of the four variants corresponded to an overall biphasic decrease in fluorescence. This is the reverse of that observed in our previous study using plasma derived protein and other recombinant mutants (23Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The difference results from the redox state of Cys-232, which greatly affects the fluorescence signal from polymerization but does not affect the polymerization process itself (unpublished observations). The close proximity of Cys-232 to one of the two tryptophans, Trp-238 (6.5 Å), and the substitution of Cys-232 for a serine in the S121C, D159C, and I360C variants are likely to change the signal. Measurements of the changes in 5-TMRIA fluorescence during polymerization of each variant at 45 °C show that in three of the four variants the derivatization process does not alter the rate of polymerization. However the S121C mutation reduced the polymerization rate by 80%, which suggests that the region around residue 121 is important in the polymerization process.Table IRate of polymerization of the α1-antitrypsin Pittsburgh variants during incubation at 0.1 mg/ml and 45 °CMeasurementRateS121CD159CPittsburgh wild typeI360CWildtype antitrypsins −1Unlabeled intrinsic fluorescence2.62 ± 0.34 × 10−52.37 ± 0.41 × 10−51.43 ± 0.33 × 10−53.65 ± 0.25 × 10−53.65 ± 0.20 × 10−55-TMRIA fluorescence0.51 ± 0.25 × 10−52.10 ± 0.57 × 10−53.17 ± 0.58 × 10−52.85 ± 0.34 × 10−52.70 ± 0.03 × 10−5Rates were obtained by fitting the data to either a double (intrinsic fluorescence) or single exponential function (5-TMRIA) (23Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Data are shown as the means ± S.D. (n = 3). The values for recombinant wild type protein are taken from Dafforn et al.(23Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Open table in a new tab Rates were obtained by fitting the data to either a double (intrinsic fluorescence) or single exponential function (5-TMRIA) (23Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Data are shown as the means ± S.D. (n = 3). The values for recombinant wild type protein are taken from Dafforn et al.(23Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). We used the optical phenomenon of fluorescence resonance energy transfer to investigate the orientation of monomeric units within the α1-antitrypsin polymer. α1-Antitrypsin was labeled with fluorophores via cysteines at four locations throughout the protein. These residues were located 1) on the reactive center loop at residue 360, 2) on the C-sheet at residue 232, 3) at the bottom of s2A at residue 121, and 4) on the top of helix F at residue 159 (Fig.1 A). Mixtures were then prepared using protein labeled at 360 and 159 with 5-TMRIA to act as FRET acceptors and protein labeled at 121, 159, 232, and 360 with 5-IAF to act as FRET donors. These mixtures were then polymerized by incubation at 45 °C for 48 h. To ensure that no monomeric material remained in the sample, FRET efficiencies were also measured following incubation at 55 °C for 48 h. The results from this experiment were identical to those carried out at 45 °C. The efficiency of FRET was measured by comparing emission spectra derived from exciting at the FRET donor wavelength of polymerized material with and without an acceptor (Fig. 1 B). Fluorescence anisotropy measurements were also made for each labeled protein to assess whether the labels showed an isotropic distribution and hence that the assumption of a value of 23 for κ 2 was valid. The values obtained ranged from 0.08 for the label on residue 232 to 0.26 for the label on residue 121. Assessment of the effect of the lack of isotropy on the value of R 0 was carried out using the methods of Dale et al. (34Dale R.E. Eisinger J. Blumberg W.E. Biophys. J. 1979; 26: 161-194Abstract Full Text PDF PubMed Scopus (648) Google Scholar) and showed that this would affect the accuracy of R 0 by 1.1 Å at most. These data were then used in combination with the respective R 0 to calculate the distances between the labels within the polymer (TableII). The FRET efficiencies for polymerized α1-antitrypsin were well within the region that allowed accurate values for R to be determined for all FRET pairs except 121–159. The measurement of FRET between proteins labeled at 159 and 360 in both directions (159 donor to 360 acceptor and 360 donor to 159 acceptor) provided an internal check of the consistency of FRET measurements. The FRET efficiencies calculated from these 2 pairings were in very close agreement (159 → 360, E = 0.53; 360 → 159, E = 0.47), providing confidence in the accuracy of the measurements.Table IIComparison of FRET efficiencies with those calculated from modeled structuresFRET pairExperimental FRET signalFRET signal calculated from model structuresOriginal A-sheet polymer (2)C-sheet polymer (32)Strand s7A A-sheet polymer (5)12121–3600.55>0.900.100.780.550.57159–3600.53>0.900.30>0.900.830.90232–3600.140.15>0.900.470.180.18360–3600.170.09>0.900.790.120.21121–15900.100.080.080.010.03159–1590.140.340.120.120.030.06232–1590.90>0.90>0.900.640.850.95360–1590.47R values were calculated with respect to a κ2value of 2/3. R 0 values were calculated for each FRET pair with respect to the differing quantum yields of the donor fluorophore. Calculation of values of theoretical FRET efficiencies were made by measuring the interfluorophore distances for each FRET pairing in each model. The values were obtained for each possible transfer within a 7-mer polymer, and the theoretical FRET efficiency was calculated by the method of Miki and Iio (31Miki M. Iio T. Biochim. Biophys. Acta. 1984; 790: 201-207Crossref PubMed Scopus (11) Google Scholar). Efficiencies calculated from models that are within 0.05 of those measured experimentally are highlighted in bold type. 1 and 2 are representative examples of structures belonging to the two structural groups found during the conformational search procedure. Open table in a new tab R values were calculated with respect to a κ2value of 2/3. R 0 values were calculated for each FRET pair with respect to the differing quantum yields of the donor fluorophore. Calculation of values of theoretical FRET efficiencies were made by measuring the interfluorophore distances for each FRET pairing in each model. The values were obtained for each possible transfer within a 7-mer polymer, and the theoretical FRET efficiency was calculated by the method of Miki and Iio (31Miki M. Iio T. Biochim. Biophys. Acta. 1984; 790: 201-207Crossref PubMed Scopus (11) Google Scholar). Efficiencies calculated from models that are within 0.05 of those measured experimentally are highlighted in bold type. 1 and 2 are representative examples of structures belonging to the two structural groups found during the conformational search procedure. Stern-Volmer constants were derived from the linear slopes of fluorescence plotted against increasing acrylamide concentration. In each case the linear fit to the experimental data was achieved with a correlation coefficient (r) of greater than 0.98, thus indicating the presence of only collision quenching (27Sherwin, S. S., and Leavis, P. C. Methods Enzymol., XLIX, 222–236.Google Scholar, 28Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1999: 237-265Crossref Google Scholar, 29Eftink M.R. Ghiron C.A. Anal. Biochem. 1981; 114: 199-227Crossref PubMed Scopus (1608) Google Scholar). Stern-Volmer constants were measured for each labeled mutant before and after polymerization (TableIII and Fig.2). These measurements allowed the change in solvent accessibility of the probe to be assessed during polymer formation. It might be expected that the transition of a protein from monomer to polymer must occlude at least some of the protein surface from solvent. Our measurements of Stern-Volmer constants for each labeled protein showed that in none of the four cases didk sv decrease substantially upon transition to the polymeric form. A decrease in k sv might have been predicted if the probe had become less solvent accessible because of the binding of a neighboring monomer in the polymer. In fact in two cases, following labeling at residues 121 and 360, thek sv increased, which is indicative of the probe becoming more accessible to solvent.Table IIIComparison of Stern-Volmer values calculated from acrylamide quenching experimentsProteinStern-Volmer constantsMonomericPolymericM −1S121C0.350.56D159C0.410.48Pittsburgh wild type (232C)0.540.59I360C0.260.50Stern-Volmer values were calculated for each labeled protein in the monomeric and polymeric form and represent an average of three experiments. Open table in a new tab Stern-Volmer values were calculated for each labeled protein in the monomeric and polymeric form and represent an average of three experiments. Three models were used in the initial assignment of FRET distances (Fig.3 A). These models represented the proposed A- and C-sheet linkages (2Elliott P.R. Lomas D.A. Carrell R.W. Abrahams J.-P. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (239) Google Scholar, 32Chang W.-S.W. Whisstock J. Hopkins P.C.R. Lesk A.M. Carrell R.W. Wardell M.R. Protein Sci. 1997; 6: 89-98Crossref PubMed Scopus (72) Google Scholar) and the s7A linkage (5Sharp A.M. Stein P.E. Pannu N.S. Carrell R.W. Berkenpas M.B. Ginsburg D. Lawrence D.A. Read R.J. Structure. 1999; 7: 111-118Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Measurements were made between fluorophores in all possible α1-antitrypsin molecules inhabiting three positions within the polymer either side of the monomer containing the donor fluorophore. This allowed for the possibility that helical arrangements of the polymers may bring nonconsecutive units into close contact (like the 1–4 interaction observed between amino acids in an α-helix). Comparison of the values for E determined by FRET and those calculated from the three existing models show distinct differences in each case. For the A-sheet model, the values for the 121–360 and 159–360 pair show significant differences, although there is good agreement with all the values in which 159 acted as acceptor. In contrast the C-sheet model has discrepancies with the transfer efficiency values for all pairings apart from 232–159 and 159–159. Conventional modeling techniques that have been employed in studies of the serpin-protease complex using FRET are not applicable to polymers. The FRET signal produced by a polymeric structure is complicated by the large number of possible positions that donors and acceptors can occupy. This problem can only be solved if the polymer forms a regular extended three-dimensional structure in which FRET only occurs between near neighbors in the polymer. Electron micrographs of serpin polymers show this to be the case (11Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar, 12Lomas D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar). This greatly simplifies the situation allowing a solution to be determined by computation. For example, an acceptor molecule on monomer nin an extended polymer may participate in FRET with a donor on then + 1 monomer as well as n + 2, n + 3, etc., and n − 1, n − 2. . . . . We have developed a computational method that automates this analysis. The program CharmM was used to develop an algorithm that performs an exhaustive search of possible conformations of α1-antitrypsin polymers. Calculations of theoretical efficiencies for transfers from n − 3 to n+ 3 were then made for each new conformation, and a total efficiency for the conformation was calculated. This allowed us to test almost 3 million conformations. A heptameric model of the serpin polymer used as initial modeling studies showed that even in the most compact polymer, the FRET from monomers more distant than n + 3 andn − 3 was insignificant. Results from this search show that the parameters imposed by the experimental FRET results were only matched by 11 polymer structures. Closer examination of these structures showed that three could be discarded because they were represented by a polymer of monomers that were spaced too far apart to allow intermolecular linkage. Two structures were discarded as the monomeric units showed extensive overlap. The six remaining structures are shown in Fig. 3 B. In all cases the monomers are arranged such that the RCL of one monomer is in close proximity to the A-sheet of a second monomer. Closer examination of the structures shows that these six candidates can be further classified into two groups: Group 1 contains five structures in which the orientation of the monomers places the RCL of one monomer close to the s7A-s4A position, and Group 2 contains one structure in which the orientation of the monomers places the RCL of one monomer close to the s2A-s3A position. To simplify the analysis of these structures, a representative of each group was analyzed in more detail in Table II. These structures have five of seven FRET efficiency values within 0.05 of those determined by experiment. The other two anomalous FRET efficiency values were measured using experimental values determined using fluorescein attached to residue 159. This may suggest that the position of 159 in the crystal structure of the monomer used to build the polymers is different to the position of 159 in the polymer. Stern-Volmer quenching constants were used to discriminate between s4A and s7A linkage. Perhaps unexpectedly there was no decrease in the solvent accessibility of any of the probes. However, closer examination of the positions of the labeled residues showed that none of them became buried in any of the polymer models. Measurement of the Stern-Volmer constant for protein labeled at position 121 showed an increase in solvent accessibility in the polymeric form. Examination of the positions of this residue in structures of α1-antitrypsin with and without insertions into the s4A position showed that upon insertion into s4A residue 121 becomes 25% more exposed. This demonstrates that the polymer must result from s4A linkage. In summary the measurement of FRET using probes at different points within α1-antitrypsin has allowed an assessment of the viability of all possible structures of the α1-antitrypsin polymer. The results of this search demonstrate that candidate structures cluster into two groups. The use of Stern-Volmer quenching experiments have allowed us to propose that the linkage is by an insertion of the reactive loop of one molecule into s4A of a second. Understanding this linkage provides a firm basis for rational drug design to block reactive center loop-β-sheet A interactions and so ameliorate the associated disease. We thank Dr. E. Stratikos (University of Illinois, Chicago, IL) for making the plasmids, Prof. Randy Read (University of Cambridge, Cambridge, UK) for helpful discussions on computational methods, and Dr. Alan Weeds (Laboratory of Molecular Biology, Cambridge, UK) for the use of the polarimeter." @default.
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• W1583012020 title "Pathogenic α1-Antitrypsin Polymers Are Formed by Reactive Loop-β-Sheet A Linkage" @default.
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