Biochemical documentation of allelic variation of the I-E antigens of the d,k, p,r, and u haplotypes

The extent of allelic variation of the E and E polypeptide chains of the I-E antigens from the H-2> ^d ,H-2 ^k , H-2 ^p , H-2 ^r , and H-2 ^u haplotypes is described. E and E chains were individually labeled with arginine or lysine and compared by tryptic peptide analysis. The results indicate minimum variability among the E polypeptides encoded by the d, k, p, and r haplotypes. However, the E ^u chain differed significantly from the other allelic E gene products. On the other hand, the E alleles demonstrated substantial variability with the E ^d being notably less similar to the other alleles than they are to each other. These findings are consistent with a number of observations regarding the serology and functions of the I-E antigens.Abbreviations MHC major histocompatibility complex - NMS normal mouse serum - NP-40 Nonidet P-40 - NTS 0.25% NP-40, 10 mM Tris-Cl, 0.15 M NaCl (pH 7.4) - SDS sodium dodecylsulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS     

The expression of I-Ed molecules in F1 hybrid mice detected with antigen-specific,I-Ed-restricted cloned T-cell lines

The expression of polymorphic determinants on I-E molecules is largely dependent on allelic variation in the E chain. We have previously analyzed the expression of E ^k and E ^b chains in F₁ hybrid mice by a combination of techniques, and have shown that functional variation detected by the responsiveness of cloned T-cell lines specific for these molecules correlates well with serological determination of E expression. In the present study, we have extended our analysis to E ^d expression in F₁ hybrid mice. We show that E ^d is relatively poorly expressed in three F₁ combinations: H-2 ^d× H-2 ^b, H-2 ^d× H-2 ^s, and H-2 ^d× H-2 ^u. The former two crosses express E chains from the H-2 ^dparent only; when recombinant strains carrying E ^b or E ^s and an active E gene are used, E ^d expression is significantly increased. On the other hand, H-2 ^umice synthesize E chains; the poor expression of E ^d chains in this F₁ hybrid apparently reflects the strong preferential association of E ^u chains with all E molecules thus far analyzed. These results confirm that E chains compete for binding to E chains and that preferential association of different allelic forms of E chains with E chains is a generalized phenomenon. They also illustrate the importance of the rate of biosynthesis of Ia chains for cell-surface expression.     

The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO and DR genes, at least three DR genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ ^{1 1} to DQ ^{2 4}, possessed a single DQ and a single DQ gene whereas both these genes were duplicated in eight other haplotypes, DQ ^{3 5} to DQ ^{9 12}. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ DQ , DR DR , and DO restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.     

Assembly-dependent expression of antigenic epitopes by β2-microglobulinb

In this report we provide evidence for the expression of antigenic epitopes on mouse (₂-microglobulin^b _2m ^b) that result from assembly with cognate H-2 class I heavy chains. For the cell line 69.9.15 (₂m^a × ₂m^b), which expresses a mutant cytosolic form of H-2K^b and wild-type H-2D^b, flow cytometry with rabbit antiserum against mouse ₂m displayed ₂m expression by cells grown in the presence or absence of fetal calf serum. By contrast, the epitopes identified by the ₂m^b-specific monoclonal antibody (mAb) S19.8 and clone 23 were not expressed by 69.9.15 cells grown in serum-containing conditions, and although S19.8 reactivity was weakly recovered by culture in the absence of serum, no such reacitivity was observed with clone 23. Strong expression of these epitopes was achieved following transfection of 69.9.15 cells with the wild-type H-2K ^b gene, indicating that the ₂m^b epitopes defined by mAb S19.8 and clone 23 were expressed when ₂m^b was assembled with an appropriate heavy chain. In support of this conclusion, we observed the recovery of the S19.8 and clone 23 epitopes by in vitro assembly of H-2K^b heavy chains with ₂m^b in the presence of the VSV N protein p52–59; however, such epitopes were expressed neither by ₂m^b prior to heterodimer assembly nor by non-conformed ₂m^b present in tissue culture supernatants recovered from H-2 class I surface positive cells. Taken together, these data indicate that in addition to the property of ₂m to modify the antigenicity of the MHC class I heavy chains, ₂m epitopes are induced in a reciprocal manner by assembly with MHC class I heavy chain molecules. Correspondence to: R. A. Zeff.     

Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity

In preparation for functional analyses, a study of the binding of H-2K^b-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2^b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2K ^bm mutant cells and on FOR-treated H-2K^b cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2KP^b-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2^b cells as indicated by cold target inhibition studies. The H-2K^b-specific CTL were probably reactive with conformational determinants of H-2K^b, which are dependent on the integrity of both the 1 and the 2 domains of the H-2K^b molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the 1 domain without affecting conformational-type CTL determinants.Abbreviations used in this paper CTL cytotoxic T lymphocyte - FOR formaldehyde - PBS phosphatebuffered saline - FCS fetal calf serum - mAb monoclonal antibody - TNBS trinitrobenzene sulfonate     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Rates of decay for the survival probability of a mutant gene

If qk is the extinction probability of a slightly supercritical branching process with offspring distribution P _kr : r = 0, 1, 2,..., then it is shown that if sup _r r ³ p _kr , < , inf ² _k > 0, and m _k 1, then 1 – q _k 2(m _k –1) _k ^–2, where m _k = _r rp _kr , ² _k = ^k _r r ² p _kr – m _k ². This provides a simple set of sufficient conditions for the validity of a conjecture of Ewens (1969) for the survival probability of a slightly advantageous mutant gene.Research supported in part by NSF grants DMS-8803639 and DMS-9007182     

One-bond carbon-proton coupling constants: Angular dependence in β-linked oligosaccharides

Summary The angular dependence of¹J_C,H in model compounds related to -linked oligosaccharides has been established by FPT INDO quantum chemical calculations. Values calculated for models of (1 1)-, (1 2)-, (1 3)- and (1 4)-linked disaccharides were compared, and the effect of the orientation of HO-2 elucidated. The angular dependence of¹J_C,H on the torsional angles ^H and ^H and the solvent dielectric constant (s) was characterized in the form:¹J_C,H = A cos2+B cos + C sin2 + D since + E + Fe. The¹J_C,H values, measured by DEPT methods for C-1-H-1 and C-X-H-X in cellobiose, cyclic trisaccharide and hexopyranoses were used to adjust the calculated angular dependences. Based on the occurrence of the conformers for agarobiose, neoagarobiose, mannobiose and methyl -xylobioside, the thermodynamically averaged <¹J_C,H > values were calculated. The results obtained (<¹J_C-1,H-1 > 162.4, <¹J_{C-4, H-4} > 147.6 Hz for methyl -xylobioside; <¹J_C-1,H-1 > 162.4 and <¹J_C-4,H-4] > 147.6 Hz for mannobiose; <¹J_C-1,H-1 > 162.8 Hz for neo agarobiose and <¹J_C-1,H-1 > 163.2 Hz for agarobiose) agree well with the experimental values of 162.7, 147.5, 160.4, 147.2, 160.9 and 165.7 Hz, respectively.     

The α2 cDNA sequence of human haptoglobin carries a bacterial promoter functionalin vivo

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete ^2FS precursor protein or only for the subunit have been placed under the control of the P_R promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the P_R promoter, the complete ^2FS constructions constitutively express a smaller polypeptide of 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (²P_F and ²P_S) located in the duplicated ^2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp² cDNA sequence, when fused upstream to the cDNA coding for ¹-antitrypsin, constitutively promotesin vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against ¹-antitrypsin or haptoglobin.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Autotrophic carbon sources for heterotrophic bacterioplankton in a floodplain lake of central Amazon

The relative contribution of autotrophic carbon sources (aquatic macrophytes, flooded forest, phytoplankton) for heterotrophic bacterioplankton was evaluated in a floodplain lake of the Central Amazon. Stable carbon isotopes (¹³C) were used as tracers. Values of ¹³C of different autotrophic sources were compared to those of dissolved organic carbon (DOC) and those of bacterially produced CO₂.The percentage of carbon derived from C₄ macrophytes for bacterially produced CO₂ was the highest, on average 89%. The average ¹³C value of CO₂ from bacterial respiration was –18.5 ± 3.3. Considering a fractionation of CO₂ of 3 by bacterial respiration, ¹³C value was –15.5, near C₄ macrophyte ¹³C value (–13.1).The average value of total DOC ¹³C was –26.8 ± 2.4. The percentage of C₄ macrophytes carbon for total DOC was on average 17%. Considering that bacteria consume mainly carbon from macrophytes, the dominance of C₃ plants for total DOC probably reflects a faster consumption of the former source, rather than a major contribution of the latter source.Heterotrophic bacterioplankton in the floodplain may be an important link in the aquatic food web, transferring the carbon from C₄ macrophytes to the consumers.     

Soluble Expression and Affinity Purification of Functional Domain of Human Acetylcholine Receptor <Emphasis Type="Italic">α</Emphasis>-subunit by the Modulation of Maltose Binding Protein

An open reading frame of the -subunit 1-205 residues (₂₀₅) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR₂₀₅ as the template and inserted into vector pMAL-c2X. The constructed pMAR₂₀₅ was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR₂₀₅ protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR₂₀₅ could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR₂₀₅ and AChR₂₀₅ were similar to that of AChR -subunit from Torpedo.Revisions received 23 September 2004     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

T-cell receptor-specific monoclonal antibodies against a V β 11-positive mouse T-cell clone

Two monoclonal antibodies specific for the mouse T-cell receptor (Tcr) have been established by immunization with a V 11⁺ T-cell clone, clone C6. One is a rat antibody, KT11 (IgG2b, k), specific for the V chain of C6, V 11. This was demonstrated by the fact that the strain distribution pattern of KT11⁺ cells was similar to that of V 5, 8, 9, 11, 12, and 13 and that the gene that encodes the molecule detected by KT11 was closely linked to V 8 in (B10 × SJL)F₁ × SJL backcross mice. Furthermore, V of C6 has been cloned from a gt10 cDNA library and was demonstrated to be identical to the V 11 published sequences. All strains of mice that do not express major histocompatibility complex class II E molecules had higher numbers of KT11^– cells than E⁺ strains. The KT11⁺ population in A strain mice and its H-2 congenic strains, however, was not affected by the presence or absence of E molecules. The other is a mouse antibody, KTL2 (IgM), specific for the idiotope of the Tcr expressed on the clone C6. Both antibodies were mitogenic and induced cytotoxicity. Expression of epitopes detected by KT11 or KTL2 was down-modulated by a T3-specific antibody 145-2C11.     

Structure function analysis of theH-2 Ab p gene

The gene encoding the H-2A ^p class II chain was isolated from a B10.P genomic library and sequenced. This gene was also used to construct transfectants of the CH12 lymphoma clone CH12.LX, which express the Ab^p gene product in association with the endogenous A ^k chain. We present here the first report of the complete nucleotide coding sequence of Abp. The predicted amino acid sequence of Abp reveals only five residues different from Ab q, four of which are present in the mature peptide. These four amino acid changes could account for the differential susceptibility of H-2 q vs H-2 p mice to the development of collagen-induced arthritis (CIA). Antibodies specific for the transfected Abp protein induce CH 12. LX cells to secrete immunoglobulin in the presence of antigen. Comparison of the amino acid sequence with other A chains that have been tested in signal transduction experiments suggests that amino acid 9 may be important to the signaling ability of class II A molecules.     

E infα supk transgene in B10 mice suppresses the development of myasthenia gravis

Mice bearing the H-2 ^bhaplotype are susceptible to the development of experimental autoimmune myasthenia gravis (EAMG), induced by acetylcholine receptor (AChR) autoimmunity. One of the genes influencing EAMG susceptibility has been mapped to the A ^blocus of the major histocompatibility complex, and the A chain has been implicated in the pathogenesis. Mice of the H-2 ^bhaplotype, including C57BL/10 (B10), have a genomic deletion of the E gene and therefore fail to express the E molecule on their cell surface. To test the hypothesis that failure to express the cell surface E molecule in B10 mice contributes to EAMG pathogenesis, E _inf ^supk transgenic B10 mice expressing the T molecule were examined. Expression of the E molecule in E _inf ^supk transgenic B10 mice partially prevented the development of EAMG.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.