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J Balbach

Publications and source records attributed to J Balbach.

12 recordsLinked to original sources
PubMed2000

Cooperativity of a protein folding reaction probed at multiple chain positions by real-time 2D NMR spectroscopy.

The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slow reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was determined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D (15)N-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that the intermediate is as compact as the native protein with many native chemical shifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates that this cooperative refolding reaction is fully synchronized. The stability of the folding intermediate was determined from the protection factors of 45 amide protons derived from a competition between refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet-native regions.

Guanidine↗
PubMed1999

Rapid collapse and slow structural reorganisation during the refolding of bovine alpha-lactalbumin.

The refolding of bovine alpha-lactalbumin (BLA) from its chemically denatured state in 6 M GuHCl has been investigated by a variety of complementary biophysical approaches. CD experiments indicate that the species formed in the early stages of refolding of the apo-protein have at least 85 % of the alpha-helical content of the native state, and kinetic NMR experiments show that they possess near-native compactness. Hydrogen exchange measurements using mass spectrometry and NMR indicate that persistent structure in these transient species is located predominantly in the alpha-domain of the native protein and is similar to that present in the partially folded A-state formed by the protein at low pH. The extent of the exchange protection is, however, small, and there is no evidence for the existence of well-defined discrete kinetic intermediates of the type populated in the refolding of the structurally homologous c-type lysozymes. Rather, both mass spectrometric and NMR data indicate that the rate-determining transition from the compact partially structured (molten globule) species to the native state is highly cooperative. The data show that folding in the presence of Ca2+ is similar to that in its absence, although the rate is increased by more than two orders of magnitude. Sequential mixing experiments monitored by fluorescence spectroscopy indicate that this slower folding is not the result of the accumulation of kinetically trapped species. Rather, the data are consistent with a model in which binding of Ca2+ stabilizes native-like contacts in the partially folded species and reduces the barriers for the conversion of the protein to its native state. Taken together the results indicate that folding of BLA, in the presence of its four disulphide bonds, corresponds to one of the limiting cases of protein folding in which rapid collapse to a globule with a native-like fold is followed by a search for native-like side-chain contacts that enable efficient conversion to the close packed native structure.

Animals↗
PubMed1999

A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy.

The rate-limiting step during the refolding of S54G/P55N ribonuclease T1 is determined by the slow trans-->cis prolyl isomerisation of Pro39. We investigated the refolding of this variant by one-dimensional (1D) and two-dimensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10 degreesC. Two intermediates could be resolved with the 1D approach. The minor intermediate, which is only present early during refolding, is largely unfolded. The major intermediate, with an incorrect trans Pro39 peptide bond, is highly structured with 33 amide protons showing native chemical shifts and native NOE patterns. They could be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spectroscopy) by using a new assignment strategy to generate positive and negative signal intensities for native and non-native NOE cross-peaks, respectively. Surprisingly, amide protons with non-native environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, including the C-terminal part of the alpha-helix, beta-strands 3 and 4 and several loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal beta-strands 1 and 2 and the C terminus (connected by the disulfide bonds), the N-terminal part of the alpha-helix, and the loops between beta-strands 4/5 and 5/6. Implications of these native and non-native structure elements of the intermediate for the refolding of S54G/P55N ribonuclease T1 and for cis/trans isomerizations are discussed.

Magnetic Resonance Spectroscopy↗
PubMed1998

Structure and dynamic properties of the single disulfide-deficient alpha-amylase inhibitor [C45A/C73A]tendamistat: an NMR study.

Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the alpha-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by 13C(alpha) heteronuclear NOE experiments with carbon in natural abundance. 15N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The 15N resonance assignment was achieved by a 15N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the beta-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel beta-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat.

Disulfides↗
PubMed1997

Detection of residue contacts in a protein folding intermediate.

Protein folding can be described in terms of the development of specific contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with alpha-lactalbumin reveal the existence of extensive NOEs between aromatic and aliphatic protons in the archetypal molten globule formed by this protein at low pH. Analysis of their time development provides direct evidence for near-native compactness of this state. Through a rapid refolding procedure the NOE intensity can be transferred efficiently into the resolved and assigned spectrum of the native state. This demonstrates the viability of using this approach to map out time-averaged interactions between residues in a partially folded protein.

Animals↗
PubMed1997

Diffusion control in an elementary protein folding reaction.

The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N &lrharr; U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose. The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together. It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding. Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy. We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein. This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior.

Bacillus subtilis↗
PubMed1996

Protein folding monitored at individual residues during a two-dimensional NMR experiment.

An approach is described to monitor directly at the level of individual residues the formation of structure during protein folding. A two-dimensional heteronuclear nuclear magnetic resonance (NMR) spectrum was recorded after the rapid initiation of the refolding of a protein labeled with nitrogen-15. The intensities and line shapes of the cross peaks in the spectrum reflected the kinetic time course of the folding events that occurred during the spectral accumulation. The method was used to demonstrate the cooperative nature of the acquisition of the native main chain fold of apo bovine alpha-lactalbumin. The general approach, however, should be applicable to the investigation of a wide range of chemical reactions.

Circular Dichroism↗
PubMed1995

Following protein folding in real time using NMR spectroscopy.

The refolding of apo bovine alpha-lactalbumin has been monitored in real time by NMR spectroscopy following rapid in situ dilution of a chemically denatured state. By examining individual resonances in the time-resolved NMR spectra, the native state has been shown to emerge in a cooperative manner from an intermediate formed in the dead-time of the experiments. The kinetics of folding to the native state are closely similar to those observed by stopped-flow fluorescence and near-UV circular dichroism. The NMR spectrum of the transient intermediate resembles closely that of the well characterized stable molten globule state formed at low pH. The results suggest that NMR can play a key role in describing at an atomic level the structural transitions occurring during protein folding.

Circular Dichroism↗
PubMed1994

Mannose transporter of Escherichia coli. Backbone assignments and secondary structure of the IIA domain of the IIABMan subunit.

The mannose transporter of Escherichia coli consists of two transmembrane and one peripheral protein subunit. The complex acts by a mechanism which couples translocation of the substrate with substrate phosphorylation. The peripheral IIABMan is a homodimer. The IIABMan monomer itself contains two domains which are linked by an Ala-Pro-rich hinge and which are both transiently phosphorylated at histidyl residues. The IIA and IIB domains can be separated by limited proteolysis. The IIA domain has a dimer molecular mass of 2 x 14 kDa. Almost complete 1H, 13C, and 15N NMR assignments of the backbone resonances of IIAMan have been achieved using 3D and 4D double-and triple-resonance techniques. Secondary structure elements were derived from NOE data. The IIA domain consists of a central beta-sheet of four parallel and one antiparallel strand (strand order 5 4 3 1 2) with helices on both sides of the sheet. The active-site His-10 is located in a loop at the C-terminus of beta-strand 1. This loop and the loop after strand 3 are at the topological switch point of the sheet.

Amino Acid Sequence↗
PubMed1994

Predicted topology of the N-terminal domain of the hydrophilic subunit of the mannose transporter of Escherichia coli.

A folding topology for the homodimeric N-terminal domain (IIA, 2 x 14 kDa) of the hydrophilic subunit (IIABman) of the mannose transporter of E. coli is proposed. The prediction is based on (i) tertiary structure prediction methods, and (ii) functional properties of site-directed mutants in correlation with NMR-derived alpha/beta secondary structure data. The 3D structure profile suggested that the overall fold of IIA is similar to that of the unrelated protein, flavodoxin, which is an open-stranded parallel beta-sheet with a strand order of 5 4 3 1 2. The 3D model of IIA, constructed using the known atomic structure of flavodoxin, is consistent with the results from site-directed mutagenesis. Recently NMR results confirmed the open parallel beta-sheet with a strand order of 4 3 1 2 (residues 1-120) of our model whereas beta-strand 5 (residues 127-130) was shown to be antiparallel to beta-strand 4. The correctly predicted fold includes 90% of the monomeric subunit sequence and contains all functional sites of the IIA domain.

Amino Acid Sequence↗
PubMed1984

[Cor triatriatum sinistrum in adults (case report)].

A case is presented of cor triatriatum sinistrum , which has been diagnosed in an adult. The findings of invasive and noninvasive studies have resulted in a diagnosis, which can be confirmed by using two-dimensional echocardiography.

Adult↗