Solution structure, dimerization, and dynamics of a lipophilic alpha/3(10)-helical, C alpha-methylated peptide. Implications for folding of membrane proteins.

The solution structure and the dimerization behavior of the lipophilic, highly C(alpha)-methylated model peptide, mBrBz-Iva(1)-Val(2)-Iva(3)-(alphaMe)Val(4)-(alphaMe)Phe(5)-(alphaMe)Val(6)-Iva(7)-NHMe, was studied by NMR spectroscopy and molecular dynamics simulations. The conformational analysis resulted in a right-handed 3(10)/alpha-helical equilibrium fast on the NMR time scale with a slight preference for the alpha-helical conformation. The NOESY spectrum showed intermolecular NOEs due to an aggregation of the heptapeptide. In addition, temperature-dependent diffusion measurements were performed to calculate the hydrodynamic radius. All these findings are consistent with an antiparallel side-by-side dimerization. The structure of the dimeric peptide was calculated with a simulated annealing strategy. The lipophilic dimer is held together by favorable van der Waals interactions in the sense of a bulge fitting into a groove. The flexibility of the helical conformations concerning an alpha/3(10)-helical equilibrium is shown in a 3 ns molecular dynamics simulation of the resulting dimeric structure. Both overall helical structures of each monomer and the antiparallel mode of dimerization are stable. However, transitions were seen of several residues from a 3(10)-helical into an alpha-helical conformation and vice versa. Hence, this peptide represents a good model in which two often-discussed aspects of hierarchical transmembrane protein folding are present: i <-- i + 3 and i <-- i + 4 local H-bonding interactions cause a specific molecular shape which is then recognized as attractive by other surrounding structures.

A new multi-quantum version of the HBHA(CBCACO)NH experiment with enhanced sensitivity for partially deuterated samples.

A new multi-quantum version of the HBHA(CBCACO)NH experiment for partially deuterated protein samples is presented. The method is based on the significant reduction of the proton and carbon relaxation rates due to multi-quantum delays in highly deuterated proteins recently published by our group. The introduction of a multi-quantum period in the coherence transfer pathway of the HBHA(CBCACO)NH experiment yields a dramatic increase of sensitivity-on average 46% with a 75% deuterated sample of the homodimeric 31 kDa E. coli IIAMan domain. Additional resolution in the proton dimension can be achieved by a double time shared approach keeping the 1H single-quantum period at a minimum.

Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein.

The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H. The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints. The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).

Automated backbone assignment of labeled proteins using the threshold accepting algorithm.

The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C beta information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A2, a protein with 124 residues.

13C-NOESY-HSQC with Split Carbon Evolution for Increased Resolution with Uniformly Labeled Proteins.

Two new pulse sequences are presented for the recording of 2D 13C-HSQC and 3D 13C-NOESY-HSQC experiments, containing two consecutive carbon evolution periods. The two periods are separated by a z-filter which creates a clean CxHz-quantum state for evolution in the second period. Each period is incremented (in a non-constant-time fashion) only to the extent that the defocusing of carbon inphase magnetization through J-coupling with neighboring carbons remains insignificant. Therefore, 13C homonuclear J-couplings are rendered ineffective, reducing the loss of signal and peak splitting commonly associated with long 13C evolution times. The two periods are incremented according to a special acquisition protocol employing a 13C-13C gradient echo to yield a data set analogous to one obtained by evolution over the added duration of both periods. The spectra recorded with the new technique on uniformly 13C-labeled proteins at twice the evolution time of the standard 13C-HSQC experiment display a nearly twofold enhancement of resolution in the carbon domain, while maintaining a good sensitivity even in the case of large proteins. Applied to the IIAMan protein of E. coli (31 kDa), the 13C-HSQC experiment recorded with a carbon evolution time of 2 x 8 ms showed a 36% decrease in linewidths compared to the standard 13C-HSQC experiment, and the S/N ratio of representative cross-peaks was reduced to 40%. This reduction reflects mostly the typical loss of intensity observed when recording with an increased resolution. The 13C-NOESY-HSQC experiment derived from the 13C-HSQC experiment yielded additional NOE restraints between resonances which previously had been unresolved. Copyright 1998 Academic Press.

A Spin System Labeled and Highly Resolved ed-H(CCO)NH-TOCSY Experiment for the Facilitated Assignment of Proton Side Chains in Partially Deuterated Samples.

The introduction of deuterated and partially deuterated protein samples has greatly facilitated the 13C assignment of larger proteins. Here we present a new version of the HC(CO)NH-TOCSY experiment, the ed-H(CCO)NH-TOCSY experiment for partially deuterated samples, introducing a multi-quantum proton evolution period. This approach removes the main relaxation source (the dipolar coupling to the directly bound 13C spin) and leads to a significant reduction of the proton and carbon relaxation rates. Thus, the indirect proton dimension can be acquired with high resolution, combined with a phase labeling of the proton resonances according to the C-C spin system topology. This editing scheme, independent of the CHn multiplicity, allows to distinguish between proton side-chain positions occurring within a narrow chemical shift range. Therefore this new experiment facilitates the assignment of the proton chemical shifts of partially deuterated samples even of high molecular weights, as demonstrated on a 31 kDa protein.

Studies on the NusB protein of Escherichia coli--expression and determination of secondary-structure elements by multinuclear NMR spectroscopy.

The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at nut (N utilization) sites of various bacterial and bacteriophage lambda genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion-exchange chromatography followed by gel-permeation chromatography. The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with 15N, 13C and 2H in various combinations. Heteronuclear three-dimensional triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on experimentally derived scalar couplings, chemical-shift values, amide-exchange data, and a semiquantitative interpretation of NOE data, the secondary structure of NusB has classified as alpha helical, comprising seven alpha helices.

Glucose transporter of Escherichia coli: NMR characterization of the phosphocysteine form of the IIB(Glc) domain and its binding interface with the IIA(Glc) subunit.

The transmembrane subunit of the glucose transporter, IICB(Glc), mediates vectorial transport with concomitant phosphorylation of glucose. Glucose phosphorylation proceeds through a cystein phosphate intermediate of the cytosolic IIB domain of IIC(Glc), which is phosphorylated by the IIA(Glc) subunit of the glucose transporter. Two- and three-dimensional NMR experiments were used to characterize the phosphorylation of the 10 kDa subclonal IIB domain and the complementary binding interfaces of [15N]IIB and [15N]IIA(Glc). The largest chemical shift perturbations and the only NOE differences accompanying IIB phosphorylation are confined to the active site residue Cys35, as well as Ile36, Thr37, Arg38, Leu39, and Arg40, which are all located in the turn between strands beta1 and beta2 and on beta2 itself. The significant increase of the amide cross-peak intensities of Ile36, Thr37, and Arg38 upon phosphorylation suggests that the conformational freedom of these groups becomes restrained, possibly due to hydrogen bonding to the oxygens of the bound phosphate and to interactions between the guanidinium group of Arg38 and the phosphoryl group. The residues of IIB which experience chemical shift perturbations upon binding of IIA are located on a protruding surface formed by residues of strands beta1, beta2, and beta4, the beta4/alpha3 loop, and residues from the first two turns of alpha3. The corresponding binding surface of the IIA(Glc) domain is comprised of residues on five adjacent beta-strands and two short helices surrounding the active site His90. The binding surface of IIA(Glc) for IIB coincides with the binding surface for HPr, the phosphoryl carrier protein by which IIA(Glc) is phosphorylated [Chen, Y., Reizer, J., Saier, M. H., Fairbrother, W. J., & Wright, P. E. (1993) Biochemistry 32, 32-37].

Secondary structure of the IIB domain of the Escherichia coli mannose transporter, a new fold in the class of alpha/beta twisted open-sheet structures.

The mannose transporter of the Escherichia coli bacterial phosphotransferase system consists of three subunits: IIAB, IIC and IID. IIABMan transfers phosphoryl groups to the transported substrate via phosphohistidine intermediates. Its IIB domain was overexpressed and isotopically labelled with 13C, 15N and 2H. Heteronuclear 3D triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on the evaluation of conformationally sensitive parameters, the secondary structure of the IIBMan domain has been determined as an alpha/beta twisted open-sheet structure consisting of a six-stranded parallel beta-sheet with the novel strand order 3-2-4-1-5-6, six helices and a short two-stranded antiparallel beta-sheet.

Solution structure of the IIB domain of the glucose transporter of Escherichia coli.

The structure of the IIBGlc domain of the Escherichia coli transporter for glucose was determined by multidimensional heteronuclear NMR. The glucose transporter (IICBGlc) belongs to the bacterial phosphotransferase system. It mediates uptake with concomittant phosphorylation of glucose. The N-terminal IICGlc domain spans the membrane, the C-terminal IIBGlc domain (residues 386-477) contains the phosphorylation site, Cys421. The structure of the subclonal IIB domain was determined based on 927 conformational constraints, including 744 NOE derived upper bounds, 43 constraints of ranges of dihedral angles based on measurements of vicinal coupling constants, and 70 upper and lower bound constraints associated with 35 hydrogen bonds. The distance geometry interpretation of the NMR data is based on the previously published sequence-specific 1H, 15N, and 13C resonance assignments [Golic Grdadolnik et al. (1994) Eur. J. Biochem. 219, 945-952]. The sequence of the secondary structure elements of IIB is alpha 1 beta 1 beta 2 alpha 2 beta 3 beta 4 alpha 3. The basic fold consists of a split alpha/beta-sandwich composed of an antiparallel sheet with strand order beta 1 beta 2 beta 4 beta 3 and three alpha-helices superimposed onto one side of the sheet. The hydrophobic helix alpha 1 is packed against helices alpha 2, alpha 3, and the beta-sheet. The phosphorylation site (Cys421) is at the end of beta 1 on the solvent-exposed face of the sheet surrounded by Asp419, Thr423 Arg424, Arg426, and Gln456 which are invariant in 15 homologous IIB domains from other PTS transporters.

Influence of serine in position i on conformation and dynamics of reverse turns.

NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cyclo(-D-Pro1-Ala2-Ser3(Bzl)-Trp4-Orn5(Z)-Tyr 6-) with and without protecting groups on Ser3 and Orn5. This peptide sequence was derived from the active loop sequence of the alpha-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard beta-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d6 solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent beta-turn conformation. While in the protected peptide with Ser3(Bzl) a beta II-turn is observed between Trp4 and Orn5, the deprotected compound reveals a beta I-turn in this region. The beta I-turn is stabilized by a backbone-sidechain hydrogen bond from Orn5N alpha H to Ser3O gamma. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of beta I-turns by serine in the i position through backbone-sidechain interactions.

The glucose transporter of Escherichia coli. Assignment of the 1H, 13C and 15N resonances and identification of the secondary structure of the soluble IIB domain.

The IICBGlc subunit of the Escherichia coli glucose transporter consists of two domains, the membrane-spanning IIC domain, and the hydrophilic IIB domain which contains the phosphorylation site (Cys421). A functional form of the IIB domain was over-expressed separately and isotopically labelled with 13C and 15N. A variety of 15N-edited and 13C, 15N triple-resonance NMR experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on the evaluation of conformationally sensitive parameters including NOE effects, scalar couplings and chemical shifts, the secondary structure of the IIB domain is presented. The protein is comprised of four beta-strands forming an antiparallel beta-sheet, two larger alpha-helices at the N- and C-termini and a smaller helical structure of residues 52-58.

The pseudo-beta I-turn. A new structural motif with a cis peptide bond in cyclic hexapeptides.

Synthesis and conformational analysis of three cyclic hexapeptides cyclo(-Gly1-Pro2-Phe3-Val4-Xaa5-Phe6), Xaa = Phe (I), D-Phe (II) and D-Pro (III), were carried out to examine the influence of proline on the formation of reverse turns and the dynamics of hydrophobic peptide regions. Assignment of all 1H and 13C resonances was achieved by homo- and heteronuclear 2D-NMR techniques (TOCSY, ROESY, HMQC, HMQC-TOCSY and HMBCS-270). The conformational analysis is based on interproton distances derived from ROESY spectra and homo- and heteronuclear coupling constants (E.COSY, HETLOC and HMBCS-270). For structural refinements restrained molecular dynamics (MD) simulations in vacuo and in DMSO were performed. Each peptide exhibits two conformations in DMSO solution due to cis-trans isomerism about the Gly-Pro peptide bond. Surprisingly the cis-Gly-Pro segment in the minor isomers is not involved in a beta VI-turn, but forms a turn structure with cis-Gly-Pro in the i and i + 1 positions. Although no stabilizing hydrogen bond is found in this turn, the phi- and psi-angles closely correspond to a beta I-turn [Pro2: phi(i + 1) -60 degrees, psi(i + 1) -30 degrees; Phe3: phi(i + 2) -100 degrees, psi(i + 2) -50 degrees]. Hence we call this structural element a pseudo-beta I-turn. As expected, in the dominating all-trans isomers proline occupies the i + 1 position of a standard beta I-turn. Therefore, cis-trans isomerization of the Gly1-Pro2 amide bond only induces a local conformational rearrangement, with minor structural changes in other parts of the molecule. However, the geometry of the other regions is affected by the chirality of the i + 1 amino acid for both isomers (beta I for Phe5, beta II' for D-Phe5 or D-Pro5).

1H, 13C, and 15N assignments and secondary structure of the FK506 binding protein when bound to ascomycin.

The 1H, 13C, and 15N resonances of FKBP when bound to the immunosuppressant, ascomycin, were assigned using a computer-aided analysis of heteronuclear double and triple resonance three-dimensional nmr spectra of [U-15N]FKBP/ascomycin and [U-15N,13C]FKBP/ascomycin. In addition, from a preliminary analysis of two heteronuclear four-dimensional data sets, 3JHN,H alpha coupling constants, amide exchange data, and the differences between the C alpha and C beta chemical shifts of FKBP to random coil values, the secondary structure of FKBP when bound to ascomycin was determined. The secondary structure of FKBP when bound to ascomycin in solution closely resembled the x-ray structure of the FKBP/FK506 complex but differed in some aspects from the structure of uncomplexed FKBP in solution.

NMR studies of an FK-506 analog, [U-13C]ascomycin, bound to FK-506-binding protein.

Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for [U-13C]ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons. The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the [U-13C]ascomycin/FKBP. This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506. However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A. The methods used for resonance assignment and structure calculation are presented in detail. Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket. This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.

1H, 13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site.

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two- and three-dimensional NMR experiments involving selectively 15N- and uniformly 15N- and 15N,13C-labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.

NMR studies of [U-13C]cyclosporin A bound to human cyclophilin B.

NMR data (1H and 13C chemical shifts, NOEs) on [U-13C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-13C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574-6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.