Determining the membrane topology of peptides by fluorescence quenching

Determination of the topology of peptides in membranes is important for characterizing and understanding the interactions of peptides with membranes. We describe a method that uses fluorescence quenching arising from resonance energy transfer ("FRET") for determining the topology of the tryptophan residues of peptides partitioned into phospholipid bilayer vesicles. This is accomplished through the use of a novel lyso-phospholipid quencher (lysoMC), N-(7-hydroxyl-4-methylcoumarin-3-acetyl)-1-palmitoyl-2-hydroxy-sn-gly cero-3-phosphoethanolamine. The design principle was to anchor the methylcoumarin quencher in the membrane interface by attaching it to the headgroup of lyso-phosphoethanolamine. We show that lysoMC can be incorporated readily into large unilamellar phospholipid vesicles to yield either symmetrically (both leaflets) or asymmetrically (outer leaflet only) labeled bilayers. LysoMC quenches the fluorescence of membrane-bound tryptophan by the F?rster mechanism with an apparent R(0) that is comparable to the thickness of the hydrocarbon core of a lipid bilayer (approximately 25 A). Consequently, the methylcoumarin acceptor predominantly quenches tryptophans that reside in the same monolayer as the probe. The topology of a peptide's tryptophan in membranes can be determined by comparing the quenching in symmetric and asymmetric lysoMC-labeled vesicles. Because it is essential to know that asymmetrically incorporated lysoMC remains so under all conditions, we also developed a second type of FRET experiment for assessing the rate of transbilayer diffusion (flip-flop) of lysoMC. Except in the presence of pore-forming peptides, there was no measurable flip-flop of lysoMC, indicating that asymmetric distributions of quencher are stable. We used these methods to show that N-acetyl-tryptophan-octylamide and tryptophan-octylester rapidly equilibrate across phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) bilayers, while four amphipathic model peptides remain exclusively on the outer monolayer. The topology of the amphipathic peptide melittin bound to POPC could not be determined because it induced rapid flip-flop of lysoMC. Interestingly, melittin did not induce lysoMC flip-flop in POPG vesicles and was found to remain stably on the external monolayer.     

Analysis of membrane lipids by 500 MHz 1H NMR

A nondestructive method has been developed for rapid analysis of lipid content of membrane extracts based on high field proton NMR spectroscopy. Lipid extraction is done by stepwise sonication of purified membranes into chloroform:methanol:water mixtures, and 1H spectra are recorded at 35 degrees C on final preparations consisting of at least 1 mg dried lipid solubilized in 2:1 CD3OD:CDCl3. Spectral peaks of lipid mixtures are assigned to lipid classes using a data base of standard lipid characteristic resonances derived from purified single membrane lipids and known mixtures of them. Peak intensities of characteristic peaks yield ratios of various lipids such as cholesterol:phospholipid and phosphatidylcholine:phosphatidylethanolamine, degree of unsaturation, average acyl chain length, total glycerol lipid content, and presence or absence of particular lipids, such as glycolipids or lysolipids. This procedure of membrane lipid analysis has been verified using known mixtures of purified standard lipids.     

A 1H spin echo NMR study of the HeLa tumour cell

Phosphorylcholine, phosphorylcreatine, lactate, glutathione, glycine and leucine were identified in living HeLa cells in the first study of this cell type by 1H spin echo NMR spectroscopy. The technique has the advantage that it is non-invasive, providing detailed structural information on individual species present in the cell matrix. It has been used in this case to study the rate of energy consumption following the activation of the glycolytic pathway with glucose.     

Monitoring membrane binding and insertion of peptides by two-color fluorescent label

Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.     

Identification of the C2-1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C-1H heteronuclear multiple quantum coherence method

Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75,000, [ring 2-13C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E. coli. Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring. One- and two-dimensional spectra revealed six signals out of a total of seven histidine residues in CATIII. pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed. Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass.     

Interactions between mastoparan B and the membrane studied by 1H NMR spectroscopy

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic alpha-helical structure from Lys2 to Leu14. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp9 are in close contact with methylene protons of SDS micelles. T1 relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp9 with Ala9, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp9 into the membrane, as well as the amphiphilic alpha-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Studies of the binding and structure of adrenocorticotropin peptides in membrane mimics by NMR spectroscopy and pulsed-field gradient diffusion.

The partition and structure of three adrenocorticotropic hormone peptides ACTH(1-10), ACTH(1-24), and ACTH(11-24) in water and in sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles were studied by 2D NMR and NMR gradient diffusion measurements. The diffusion rates, the NH chemical shifts, and the nuclear Overhauser effect patterns provided a coherent picture of binding of these peptides. All three peptides are significantly partitioned in the negatively charged SDS micelles and possess definite secondary structure, as opposed to random structures in water. For ACTH (1-24), the hydrophobic 1-10 segment is partitioned in DPC micelles, but the charged 11-24 segment prefers to remain in the aqueous region. ACTH(11-24) does not bind significantly to the DPC micelles. The binding of the ACTH peptides in these two widely used "membrane mimics" are substantially different from that in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers obtained by attenuated total reflection infrared spectroscopy and from our preliminary diffusion studies of the same peptides in POPC vesicles. This study showed that, in a given micellar medium, all corresponding segments of these peptides are located in the same membrane environment in the system, regardless of whether these segments exist by themselves or are attached to other segments. This result may contradict the membrane-compartments concept of Schwyzer, which suggests that ACTH(1-10) and ACTH(1-24) are located in different membrane compartments because they have different address segments, and consequently, bind to different receptors. The present results also suggest that the assumption that micelles are good membrane mimics should be carefully examined.     

Characterization of autocatalytic conversion of precursor BACE1 by heteronuclear NMR spectroscopy

Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understand the mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversion of BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to study the structural changes accompanying the autocatalytic conversion. NMR data revealed local conformational changes from a partially disordered to a well-folded conformation associated with the conversion. The conformational changes are largely concentrated in the NH(2)-terminal lobe. Conversely, the active site conformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteins were further characterized for their ability to interact with a substrate-based transition state BACE1 peptide inhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor, which is identical to the bound conformation of the mature protein. The interaction of the inhibitor with both the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale, indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does not hinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1 represents a high quality well-folded protein which is highly stable over a long period of time, and is suitable for evaluation of inhibitor binding by NMR for drug intervention.     

Two-dimensional 1H/13C heteronuclear chemical shift correlation spectroscopy of lipid bilayers.

Using solid-state magic angle spinning nuclear magnetic resonance (NMR) techniques, we have obtained two-dimensional (2D), 1H/13C chemical shift-correlated spectra of liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers in 30 wt% PO4/D2O buffer. Linewidths in both the 13C and the 1H dimensions were less than 0.3 ppm wide. The 2D spectrum consists of chemical shift correlations between all resolvable, directly bonded 13C-1H pairs and exhibits considerably greater spectral dispersion than either ID 1H or 13C MAS spectra. This approach promises to be an important tool in structural studies of biological membranes.     

Lipid composition of integral purple membrane by 1H and 31P NMR

In the purple membrane (PM) of halobacteria, lipids stabilize the trimeric arrangement of bacteriorhodopsin (BR) molecules and mediate the packing of the trimers in a regular crystalline arrangement. To date, the identification and quantification of these lipids has been based either on lipid extraction procedures or structural models. By directly solubilizing PMs from Halobacterium salinarum in aqueous detergent solutions (SDS or Triton X-100), we avoided any separation or modification steps that might modify the lipid composition or even the lipid molecules themselves. Our analysis of integral PM preparations should resolve partially conflicting literature data on the lipid composition of the PM. Using 31P and 1H NMR of detergent-solubilized but otherwise untreated samples, we found two glycolipids and 6.4 +/- 0.1 phospholipids per BR molecule, 4.4 +/- 0.1 of the latter being the phosphatidylglycerophosphate methyl ester. The only glycolipid detected was S-TGD-1. For an additional glycolipid, glycocardiolipin, that was recently identified in lipid extracts, we show that it was produced mainly during the lipid extraction procedure but also was partially dependent on the preparation of the PM suspensions.     

Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy

The backbone dynamics of uniformly 15N-labeled interleukin-1 beta are investigated by using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. 15N T1, T2, and NOE data at a spectrometer frequency of 600 MHz are obtained for 90% of the backbone amide groups. The data provide evidence for motions on three time scales. All the residues exhibit very fast motions on a time scale of approximately less than 20-50 ps that can be characterized by a single-order parameter with an average value of 0.82 +/- 0.05. For a model comprising free diffusion within a cone, these residue-specific order parameters translate to an average cone semiangle of 20.7 +/- 3.3 degrees. Thirty-two residues also display motions on a time scale of 0.5-4 ns, slightly less than the overall rotational correlation time of the protein (8.3 ns). These additional motions must be invoked to account for the discrepancy between experiment and the simplest theoretical formulation in which the internal motions are described by only two parameters, a generalized order parameter and an effective correlation time [Lipari, G., & Szabo, A. (1982a) J. Am. Chem. Soc. 104, 4546-4559]. In particular, while the simple formulation can account for the 15N T1 and T2 data, it fails to account for the 15N-1H NOE data and yields calculated values for the NOEs that are either too small or negative, whereas the observed NOEs are positive. With the introduction of two internal motions that are faster than the rotational correlation time and differ in time scales by at least 1-2 orders of magnitude [Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., & Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991], all the relaxation data for these 32 residues can be fitted by two order parameters and an effective correlation time for the slower of the two internal motions. A simple model for these two motions is one in which the very fast motion involves axially symmetric diffusion within a cone, while the slower motion comprises jumps between two different orientations of the NH vector. For such a model the jump angle (excluding the C-terminal residue) ranges from 15 degrees to 69 degrees with a mean value of 28.6 +/- 14.0 degrees. Another 42 residues are characterized by some sort of motion on the 30-ns-10-ms time scale, which results in 15N line broadening due to chemical exchange between different conformational substates with distinct 15N chemical shifts.(ABSTRACT TRUNCATED AT 400 WORDS)     

1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides

The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its Trp-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro peptide bonds. However, the orientation of the ligand in the aromatic recognition groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A., and Lim, W. A. (2000) Nat. Struct. Biol. 7, 611-613). Because the bound peptide conformation could also differ as observed for peptide ligands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here studied by solution NMR methods. First, the proton resonance perturbations on the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta-hairpin Ser(11)-Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 microm for Cdc25 and tau peptides, respectively, were found. Several intermolecular nuclear Overhauser effects between WW domain and substrates were obtained from a ligand-saturated solution and were used to determine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide ligands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.     

The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.     

Identification of the metal coordinating residues in the DNA binding domain of the glucocorticoid receptor by 113Cd-1H heteronuclear NMR spectroscopy

Two-dimensional 1H-113Cd HSQC and relay HSQC experiments were performed on the 113Cd substituted DNA binding domain of the rat glucocorticoid receptor. The results of these experiments combined with sequence-specific assignments allowed the identification of all coordinating cysteines. It was found that C495 and not C500 is the fourth coordinating cysteine in the second zinc-finger. A signal at approximately 2 ppm previously assigned to a epsilon-CH3 of a methionine residue coordinating to a third, weakly bound, cadmium ion, was identified as the C443 beta proton ligating to the metal ion in the first zinc-finger. No indications were found for the presence of a previously suggested third metal ion binding site.     

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.     

Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study

MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.     

Determination of the secondary structure and molecular topology of interleukin-1 beta by use of two- and three-dimensional heteronuclear 15N-1H NMR spectroscopy

A study of the regular secondary structure elements of recombinant human interleukin-1 beta has been carried out using NMR spectroscopy. Using a randomly 15N labeled sample, a number of heteronuclear three- and two-dimensional NMR experiments have been performed, which have enabled a complete analysis of short-, medium-, and long-range NOEs between protons of the polypeptide backbone, based on the sequence-specific resonance assignments that have been reported previously [Driscoll, P. C., Clore, G. M., Marion, D., Wingfield, P. T., & Gronenborn, A. M. (1990) Biochemistry 29, 3542-3556]. In addition, accurate measurements of a large number of 3JHN alpha coupling constants have been carried out by two-dimensional heteronuclear multiple-quantum-coherence-J spectroscopy. Amide NH solvent exchange rates have been measured by following the time dependence of the 15N-1H correlation spectrum of interleukin-1 beta on dissolving the protein in D2O solution. Analysis of these data indicate that the structure of interleukin-1 beta consists of 12 extended beta-strands aligned in a single extended network of antiparallel beta-sheet structure that in part folds into a skewed six-stranded beta-barrel. In the overall structure the beta-strands are connected by tight turns, short loops, and long loops in a manner that displays approximate pseudo-three-fold symmetry. The secondary structure analysis is discussed in the light of the unrefined X-ray structure of interleukin-1 beta at 3-A resolution [Priestle, J. P., Sch?r, H.-P., & Grütter, M. G. (1988) EMBO J. 7, 339-343], as well as biological activity data. Discernible differences between the two studies are highlighted. Finally, we have discovered conformational heterogeneity in the structure of interleukin-1 beta, which is characterized by an exchange rate that is slow on the NMR chemical shift time scale.     

Interaction of yeast iso-1-cytochrome c with cytochrome c peroxidase investigated by [15N, 1H] heteronuclear NMR spectroscopy

The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.     

Determination of the secondary structure of the DNA binding protein Ner from phage Mu using 1H homonuclear and 15N-1H heteronuclear NMR spectroscopy

The sequential resonance assignment of the 1H and 15N NMR spectra of the DNA binding protein Ner from phage Mu is presented. This is carried out by using a combination of 1H-1H and 1H-15N two-dimensional experiments. The availability of completely labeled 15N protein enabled us to record a variety of relayed heteronuclear multiple quantum coherence experiments, thereby enabling the correlation of proton-proton through-space and through-bond connectivities with the chemical shift of the directly bonded 15N atom. These heteronuclear experiments were crucial for the sequential assignment as the proton chemical shift dispersion of the Ner protein is limited and substantial overlap precluded unambiguous assignment of the homonuclear spectra in several cases. From a qualitative interpretation of the NOE data involving the NH, C alpha H, and C beta H protons, it is shown that Ner is composed of five helices extending from residues 11 to 22, 27 to 34, 38 to 45, 50 to 60, and 63 to 73.