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1.
G J Garssen C W Hilbers J G Schoenmakers J H van Boom 《European journal of biochemistry》1977,81(3):453-463
The interaction of gene V protein from bacteriophage M13 with the self-complementary tetranucleotide d(pC-G-C-G) was studied by 1H and 31P nuclear magnetic resonance. It is shown, using the hydrogen-bonded proton resonances of the Watson-Crick base pairs as a probe, that the protein is able to unwind the small double-helical fragment even at 0 degrees C. Binding of the tetranucleotide causes changes in the aromatic part of the 1H NMR spectrum of the complex, suggesting that aromatic residues, most likely tyrosines, take part in the protein.nucleic-acid interaction. From the 31P NMR spectra of the protein.nucleic-acid complex it follows that the pK value of the 5'-terminal phosphate is lower than for the free nucleic acid species. Moreover, it could be shown that the exchange of the protein between nucleic acid substrates is fast. Combination of these measurements has led us to derive a mechanism of unwinding on the tetranucleotide level. To a large extent the unwinding is determined by fluctuations in the double-helical DNA structure. 相似文献
2.
Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high-resolution crystal structure. In addition, a two-step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin. The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945A resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S.W., Gl?ckner, J. Biochemistry 20:33-37, 1981) as modified (Manavalan, P., Johnson, W.C., Jr. Anal. Biochem. 167:76-85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane-active toxins homologous to crambin (alpha 1- and beta-purothionin, phoratoxin A and B, and viscotoxin A3 and B). The new program SEQ (pronounced "seek") was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location. Both CD-arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues. 相似文献
3.
《FEBS letters》1987,219(2):426-430
Crambin displays amino acid heterogeneity at positions 22 (Pro or Ser) and 25 (Leu or Ile). Using reversed phase HPLC the crambin mixture can be resolved into two protein fractions. It is shown by amino acid analysis and NMR spectroscopy that these fractions represent single proteins (Ser-22/Ile-25 and Pro-22/ Leu-25 species). A first characterization of the 1H-NMR spectra of these species is presented. 相似文献
4.
The resonances of the aromatic rings in the 1H NMR spectra at 360 MHz of ferrocytochrome c-552 of Euglena gracilis were investigated by double resonance techniques. The spin systems of the two tryptophan and four of the tyrosine residues could be identified. This analysis of the aromatic region of the 1H NMR spectrum provided evidence that His-14 is bound to the heme iron. It gave also some insight into the molecular dynamics of ferrocytochrome c-552 in that it showed that of the six aromatic rings, four tyrosines were rotating rapidly about the Cbeta-Cgamma bond, while one tyrosine and the single phenylalanine were restricted in their rotational mobilities by their environmnent in the protein. 相似文献
5.
E A de Jong R N Konings B J Harmsen C W Prinse C W Hilbers 《European journal of biochemistry》1987,167(3):563-572
Under physiological conditions and at concentrations needed for NMR studies, severe aggregation of the gene-5 protein of the filamentous phage IKe occurs. Conditions are described for which well-resolved 1H-NMR spectra of the protein can be obtained. The aromatic part of the spectrum is analyzed by means of two-dimensional NMR techniques; a complete interpretation is presented. Oligonucleotide binding studies reveal that just one phenylalanyl residue and one tyrosyl residue are influenced by the binding of rAMP, (dA)2, (dA)3, (dA)4, (dA)6, d(pT)3 or (dT)4. Upon binding, the aromatic resonances of these amino acid residues are shifted upfield by about 0.4-0.5 ppm. NMR measurements at different pH values demonstrate that only one of the two histidyl residues is freely titratable. From CIDNP experiments it is concluded that three out of five tyrosyl residues are located at the surface of the protein. Measurements carried out as a function of protein concentration indicate the occurrence of specific protein-protein interactions between dimeric gene-5-protein molecules. The data obtained are compared with those available for the gene-5 protein of M13. It follows from the comparison that these proteins mimic each other in almost every respect. 相似文献
6.
N epsilon-[2H6]Isopropyllysyl-beta-lactoglobulin was prepared by reductive alkylation of beta-lactoglobulin with [2H6]acetone and NaBH4 to provide a 2H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein's 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Lys47 was not modified; Lys135,138,141, located along an amphipathic helical rod, were each partially unmodified. All other lysine residues were at least 90% modified. Average correlation times calculated from 2H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively, with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. 2H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of beta-lactoglobulin previously reported from 31P NMR studies of the phospholipids complexed with beta-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the X-ray structure [Papiz et al. (1986) Nature (London) 324, 383-385] supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped beta-barrel thought to contain binding sites for small lipid-soluble molecules. 相似文献
7.
P J Folkers A P Stassen J P van Duynhoven B J Harmsen R N Konings C W Hilbers 《European journal of biochemistry》1991,200(1):139-148
Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR. 相似文献
8.
C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain 总被引:10,自引:0,他引:10
Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin. 相似文献
10.
The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the 13C and 1H nuclei of the coat protein. Both the 13C and 1H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance 13C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The 1H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a 1H spectra gives the pKas for the tyrosines. 相似文献
11.
Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy 总被引:8,自引:0,他引:8
The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (153 residues, Mr = 17,400) has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. We show that the problems of amide NH and C alpha H chemical shift degeneracy that are prevalent for proteins of this size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1-interleukin 1 beta. The complete list of 15N and 1H assignments is given for all the amide NH and C alpha H resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1 beta. 相似文献
12.
Nuclear magnetic resonance and circular dichroism studies of a duplex--single-stranded hairpin loop equilibrium for the oligodeoxyribonucleotide sequence d(CGCGATTCGCG). 总被引:16,自引:15,他引:1
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M F Summers R A Byrd K A Gallo C J Samson G Zon W Egan 《Nucleic acids research》1985,13(17):6375-6386
Nuclear magnetic resonance (NMR) and circular dichroism (CD) studies have been carried out with the oligodeoxyribonucleotide mismatch sequence, d(CGCGATTCGCG), 1. It has been found that 1 exists, in solution, as an equilibrium mixture of slowly interconverting, structured conformational isomers, 1a and 1b. On the basis of the concentration dependence of the 1a-1b equilibrium, the 1H NMR spectrum of the imino protons of the nucleotide bases, and the individual CD spectra of 1a and 1b, it is suggested that the two species correspond to a B-type DNA duplex and a single-stranded, hairpin-loop structure; the portion of the single-stranded species not involved in the loop appears to have a B-type DNA structure (on the basis of the CD measurements). To facilitate 1H NMR resonance assignments, the two possible des-methyl thymidine derivatives of 1 were synthesized; the effect of this substitution on the physical chemical properties of 1 was explored. The 1H NMR spectra of 1, as a function of temperature, showed that, under conditions wherein both species were present to a significant extent, the duplex form melted at a lower temperature than the single-stranded, hairpin loop structure. 相似文献
13.
The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time. 相似文献
14.
1H NMR (500 MHz) of gene 32 protein--oligonucleotide complexes 总被引:6,自引:0,他引:6
In concentrated solutions, gene 32 single-stranded DNA binding protein from bacteriophage T4 (gene 32P) forms oligomers with long rotational correlation times, rendering 1H NMR signals from most of the protons too broad to be detected. Small flexible N- and C-terminal domains are present, however, the protons of which give rise to sharp resonances. If the C-terminal A domain (48 residues) and the N-terminal B domain (21 residues) are removed, the resultant core protein of 232 residues (gene 32P) retains high affinity for ssDNA and remains a monomer in concentrated solution, and most of the proton resonances of the core protein can now be observed. Proton NMR spectra (500 MHz) of gene 32P and its complexes with ApA, d(pA)n (n = 2, 4, 6, 8, and 10), and d(pT)8 show that the resonances of a group of aromatic protons shift upfield upon oligonucleotide binding. Proton difference spectra show that the 1H resonances of at least one Phe, one Trp, and five Tyr residues are involved in the chemical shift changes observed with nucleotide binding. The number of aromatic protons involved and the magnitude of the shifts change with the length of the oligonucleotide until the shifts are only slightly different between the complexes with d(pA)8 and d(pA)10, suggesting that the binding groove accommodates approximately eight nucleotide bases. Many of the aromatic proton NMR shifts observed on oligonucleotide complex formation are similar to those observed for oligonucleotide complex formation with gene 5P of bacteriophage fd, although more aromatic residues are involved in the case of gene 32P.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
ZFY, a male-associated Zn-finger protein encoded by the human Y chromosome, exhibits a distinctive two-finger repeat: whereas odd-numbered domains fit a general consensus, even-numbered domains exhibit systematic differences. Do these odd and even sequences encode structurally distinct surfaces for DNA recognition? As a first step toward answering this question, we have recently described the sequential 1H NMR assignment of a representative nonconsensus Zn finger (designated ZFY-6T) based on 2D NMR studies of a 30-residue peptide [Kochoyan, M., Havel, T.F., Nguyen, D.T., Dahl, C.E., Keutmann, H. T., & Weiss, M.A. (1991) Biochemistry 30, 3371-3386]. Initial structural modeling by distance geometry/simulated annealing (DG/SA) demonstrated that this peptide retained the N-terminal beta-hairpin and C-terminal alpha-helix (beta beta alpha motif) observed in consensus Zn fingers. However, the precision of this initial structure was limited by resonance overlap, which led to ambiguities in the assignment of key NOEs in the hydrophobic core. In this paper these ambiguities are resolved by selective deuterium labeling, enabling a refined structure to be calculated by DG/SA and restrained molecular dynamics. These calculations provide a detailed view of the hydrophobic core and protein surface, which are analyzed in reference to previously characterized Zn fingers. Variant (even) and consensus (odd) aromatic residues Y10 and F12, shown in an "aromatic swap" analogue to provide equivalent contributions to the hydrophobic core [Weiss, M.A., & Keutmann, H.T. (1990) Biochemistry 29, 9808-9813], nevertheless exhibit striking differences in packing interactions: Y10--but not F12--contributes to a contiguous region of the protein surface defined by putative specificity-determining residues. Alternating surface architectures may have implications for the mechanism of DNA recognition by the ZFY two-finger repeat. 相似文献
16.
P Gettins 《Biochemistry》1987,26(5):1391-1398
1H NMR has been used to characterize and compare the structures of antithrombin III from human, bovine, and porcine plasma as well as to investigate the interactions of each of these proteins with heparin fragments of defined length. The amino acid compositions of the three proteins are very similar, which is reflected in the gross features of their 1H NMR spectra. In addition, aromatic and methyl proton resonances in upfield-shifted positions appear to be common to all three proteins and suggest similar tertiary structures. Human antithrombin III has five histidine residues, bovine has six, and porcine has five. The C(2) proton from each of these residues gives a narrow resonance and titrates with pH; the pKa's are in the range 5.15-7.25. It is concluded that all histidines in each protein are surface residues with considerable independent mobility. The carbohydrate chains in each protein also give sharp resonances consistent with a surface location and motional flexibility. The 1H spectra are sensitive to heparin binding. Although heparin resonances obscure protein resonances in the region 3.2-6.0 ppm, difference spectra between antithrombin III with and without heparin show clear perturbation of a small number of aromatic and aliphatic protein protons. These resonances include those of histidine C(2) and C(4) protons, of 10-20 other aromatic protons, of a methyl group, and also of protons with chemical shifts similar to those of lysine and/or arginine side chains. For human antithrombin III, it was shown that heparin fragments 8, 10, and 16 sugar residues in length result in almost identical perturbations to the protein. In contrast, tetrasaccharide results in fewer perturbations.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Conformations of P2 Protein of Peripheral Nerve Myelin by Nuclear Magnetic Resonance Spectroscopy 总被引:1,自引:1,他引:0
High-resolution 1H NMR spectra of P2 protein from bovine peripheral nerve myelin indicate that the protein contains a high degree of tertiary structure in aqueous solution. Denaturation of the protein in urea solutions is a multi-step process. Binding of lysophosphatidylcholine micelles to the protein causes a conformational change and a broadening of NMR peaks from side chains of aromatic amino acid and methionine residues, with much less effect on upfield methyl resonances. 相似文献
18.
Phosphorus-31 nuclear magnetic resonance (NMR) studies on the two phosphorus nuclei of the phosphonium analogue (Me3P+CH2CH2OPO3(2-)) of phosphocholine are used to monitor the charged subsites in the phosphocholine-binding immunoglobulin A mouse myeloma M603. Comparison of the 270-MHz 1H NMR difference spectrum on addition of either this analogue or phosphocholine to M603 and the almost identical changes in the pKa values of the phosphate groups on binding to M603 confirm that the analogue is a good model for phosphocholine. The pKa of the phosphate groups is decreased by 0.5 unit on binding to M603, which is consistent with the phosphate group being hydrogen bonding to Tyr-33H and Arg-95L, as suggested from the X-ray structure, and also implies that the binding energies for the mono- and dianion are similar. The P+Me3 moiety is used to probe the electrostatic interactions in the choline subsite. Titration of the chemical shift of the phosphonium phosphorus reflects a group on the protein that has a pKa value of less than or equal to 5, which from the refined X-ray structure (D.R. Davies, personal communication) of the site is assigned to Asp-97L. The choline subsite is monitored by using 1H NMR difference spectra, which indicates that the subsite is highly aromatic as expected from the crystal structure that places Trp-107H and Tyr-100L in this subsite. The ring current interactions from these rings can account for the 1H NMR chemical shift data on choline. 相似文献
19.
Dimerization of lambda repressor is required for its binding to operator DNA. As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor. Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser). The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups. However, significant differences are observed in their dimerization properties. 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution. The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer. Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization. The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein. 相似文献
20.
The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1. Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system. Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3. Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions. Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer region of the three transmembrane helices could contribute to the near-UV CD spectrum through helix-helix interactions. 相似文献