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Hans Robert Kalbitzer

Publications and source records attributed to Hans Robert Kalbitzer.

At least 19 recordsLinked to original sources
PubMed2006

AUREMOL-RFAC-3D, combination of R-factors and their use for automated quality assessment of protein solution structures.

We present here the computer program AUREMOL-RFAC-3D that is a generalization of the previously published program RFAC for the fully automated estimation of residual indices (R-factors) from 2D NOESY spectra. It is part of the larger AUREMOL software package (www.auremol.de). RFAC-3D calculates R-factors directly from two-dimensional homonuclear NOESY spectra as well as from three-dimensional (15)N or (13)C edited NOESY-HSQC spectra and thus extends the application range to larger proteins. The fully automated method includes automated peak picking and integration, a Bayesian noise and artifact recognition and the use of the complete relaxation matrix formalism. To enhance the reliability of the calculated R-factors the method is also generalized to calculate combined R-factors from a set of 2D and 3D-spectra. For an optimal combination of the information derived from different sources a plausible formalism had to be derived. In addition, we present a novel direct R-factors based measure that correlates an R-factors as defined in this paper to the root mean square deviation of the actual structure from the optimal structure. The new program has been successfully tested on the histidine containing phosphocarrier protein (HPr) from Staphylococcus carnosus and on the Ras-binding domain (RBD) of the Ral guanine-nucleotide dissociation stimulation factor (RalGDS).

Bacterial Proteins↗
PubMed2006

Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy.

BACKGROUND: Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPC. The conversion and thus the propensity of PrPC to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physico-chemical properties of proteins as well as the dynamics and structure of folding intermediates. RESULTS: Conformational intermediates of the human prion protein huPrPC were characterized by a combination of hydrostatic pressure (up to 200 MPa) with two-dimensional NMR spectroscopy. All pressure effects showed to be reversible and there is virtually no difference in the overall pressure response between the folded core of the N-terminal truncated huPrPC(121-230) and the full-length huPrPC(23-230). The only significant differences in the pressure response of full-length and truncated PrP suggest that E168, H187, T192, E207, E211 and Y226 are involved in a transient interaction with the unfolded N-terminus. High-pressure NMR spectroscopy indicates that the folded core of the human prion protein occurs in two structural states N1 and N2 in solution associated with rather small differences in free enthalpies (3.0 kJ/mol). At atmospheric pressure approximately 29% of the protein are already in the pressure favored conformation N2. There is a second process representing two possible folding intermediates I1 and I2 with corresponding average free enthalpies of 10.8 and 18.6 kJ/mol. They could represent preaggregation states of the protein that coexist at ambient pressure with a very small population of approximately 1.2% and less than 0.1%. Further the pressure response of the N-terminus indicates that four different regions are in a fast equilibrium with non-random structural states whose populations are shifted by pressure. CONCLUSION: We identified pressure stabilized folding intermediates of the human prion protein. The regions reflecting most strongly the transition to the intermediate states are the beta1/alpha1-loop and the solvent exposed side of alpha3. The most pressure-sensitive region (representing mainly intermediate I1) is the loop between beta-strand 1 and alpha-helix 1 (residue 139-141), indicating that this region might be the first entry point for the infectious conformer to convert the cellular protein.

Humans↗
PubMed2006

A general method for the unbiased improvement of solution NMR structures by the use of related X-ray data, the AUREMOL-ISIC algorithm.

BACKGROUND: Rapid and accurate three-dimensional structure determination of biological macromolecules is mandatory to keep up with the vast progress made in the identification of primary sequence information. During the last few years the amount of data deposited in the protein data bank has substantially increased providing additional information for novel structure determination projects. The key question is how to combine the available database information with the experimental data of the current project ensuring that only relevant information is used and a correct structural bias is produced. For this purpose a novel fully automated algorithm based on Bayesian reasoning has been developed. It allows the combination of structural information from different sources in a consistent way to obtain high quality structures with a limited set of experimental data. The new ISIC (Intelligent Structural Information Combination) algorithm is part of the larger AUREMOL software package. RESULTS: Our new approach was successfully tested on the improvement of the solution NMR structures of the Ras-binding domain of Byr2 from Schizosaccharomyces pombe, the Ras-binding domain of RalGDS from human calculated from a limited set of NMR data, and the immunoglobulin binding domain from protein G from Streptococcus by their corresponding X-ray structures. In all test cases clearly improved structures were obtained. The largest danger in using data from other sources is a possible bias towards the added structure. In the worst case instead of a refined target structure the structure from the additional source is essentially reproduced. We could clearly show that the ISIC algorithm treats these difficulties properly. CONCLUSION: In summary, we present a novel fully automated method to combine strongly coupled knowledge from different sources. The combination with validation tools such as the calculation of NMR R-factors strengthens the impact of the method considerably since the improvement of the structures can be assessed quantitatively. The ISIC method can be applied to a large number of similar problems where the quality of the obtained three-dimensional structures is limited by the available experimental data like the improvement of large NMR structures calculated from sparse experimental data or the refinement of low resolution X-ray structures. Also structures may be refined using other available structural information such as homology models.

Algorithms↗
PubMed2006

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy.

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.

Crystallization↗
PubMed2005

NMR spectroscopic study of noble gas binding into the engineered cavity of HPr(I14A) from Staphylococcus carnosus.

Xenon binding into preexisting cavities in proteins is a well-known phenomenon. Here we investigate the interaction of helium, neon, and argon with hydrophobic cavities in proteins by NMR spectroscopy. 1H and 15N chemical shifts of the I14A mutant of the histidine-containing phosphocarrier protein (HPr(I14A)) from Staphylococcus carnosus are analyzed by chemical shift mapping. Total noble gas induced chemical shifts, Delta, are calculated and compared with the corresponding values obtained using xenon as a probe atom. This comparison reveals that the same cavity is detected with both argon and xenon. Measurements using the smaller noble gases helium and neon as probe atoms do not result in comparable effects. The dependence of amide proton and nitrogen chemical shifts on the argon concentration is investigated in the range from 10 mM up to 158 mM. The average dissociation constant for argon binding into the engineered cavity is determined to be about 90 mM.

Amino Acids↗
PubMed2005

A novel mechanism for the modulation of the Ras-effector interaction by small molecules.

When proteins require different conformations for their biological function, all these functional states have to coexist simultaneously in solution. However, the corresponding Gibbs free energy differences are usually rather high and thus the conformation with lowest energy predominates in solution whereas the populations of the states with higher energy (excited states) are very small. A stabilization of these excited states can be used as a novel principle to influence the activity of proteins by small molecules. For a proof of this principle, we selected the Ras protein that was shown by (31)P NMR spectroscopy to exist in solution in at least two different conformational states in its GTP form. One of these states shows a drastically reduced affinity to effectors. With Zn(2+)-cyclen we found a small molecule which selectively stabilizes the weak-binding state. It may serve as lead compound for the development of a new type of Ras-inhibitors.

Humans↗
PubMed2005

Solution structure of the Ran-binding domain 2 of RanBP2 and its interaction with the C terminus of Ran.

The termination of export processes from the nucleus to the cytoplasm in higher eukaryotes is mediated by binding of the small GTPase Ran as part of the export complexes to the Ran-binding domains (RanBD) of Ran-binding protein 2 (RanBP2) of the nuclear pore complex. So far, the structures of the first RanBD of RanBP2 and of RanBP1 in complexes with Ran have been known from X-ray crystallographic studies. Here we report the NMR solution structure of the uncomplexed second RanBD of RanBP2. The structure shows a pleckstrin homology (PH) fold featuring two almost orthogonal beta-sheets consisting of three and four strands and an alpha-helix sitting on top. This is in contrast to the RanBD in the crystal structure complexes in which one beta-strand is missing. That is probably due to the binding of the C-terminal alpha-helix of Ran to the RanBD in these complexes. To analyze the interaction between RanBD2 and the C terminus of Ran, NMR-titration studies with peptides comprising the six or 28 C-terminal residues of Ran were performed. While the six-residue peptide alone does not bind to RanBD2 in a specific manner, the 28-residue peptide, including the entire C-terminal helix of Ran, binds to RanBD2 in a manner analogous to the crystal structures. By solving the solution structure of the 28mer peptide alone, we confirmed that it adopts a stable alpha-helical structure like in native Ran and therefore serves as a valid model of the Ran C terminus. These results support current models that assume recognition of the transport complexes by the RanBDs through the Ran C terminus that is exposed in these complexes.

Models, Molecular↗
PubMed2005

A restraint molecular dynamics and simulated annealing approach for protein homology modeling utilizing mean angles.

BACKGROUND: We have developed the program PERMOL for semi-automated homology modeling of proteins. It is based on restrained molecular dynamics using a simulated annealing protocol in torsion angle space. As main restraints defining the optimal local geometry of the structure weighted mean dihedral angles and their standard deviations are used which are calculated with an algorithm described earlier by Doker et al. (1999, BBRC, 257, 348-350). The overall long-range contacts are established via a small number of distance restraints between atoms involved in hydrogen bonds and backbone atoms of conserved residues. Employing the restraints generated by PERMOL three-dimensional structures are obtained using standard molecular dynamics programs such as DYANA or CNS. RESULTS: To test this modeling approach it has been used for predicting the structure of the histidine-containing phosphocarrier protein HPr from E. coli and the structure of the human peroxisome proliferator activated receptor gamma (Ppar gamma). The divergence between the modeled HPr and the previously determined X-ray structure was comparable to the divergence between the X-ray structure and the published NMR structure. The modeled structure of Ppar gamma was also very close to the previously solved X-ray structure with an RMSD of 0.262 nm for the backbone atoms. CONCLUSION: In summary, we present a new method for homology modeling capable of producing high-quality structure models. An advantage of the method is that it can be used in combination with incomplete NMR data to obtain reasonable structure models in accordance with the experimental data.

Algorithms↗
PubMed2005

Conformational states of Ras complexed with the GTP analogue GppNHp or GppCH2p: implications for the interaction with effector proteins.

The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.

Amino Acid Substitution↗
PubMed2005

PERMOL: restraint-based protein homology modeling using DYANA or CNS.

SUMMARY: PERMOL is a new restraint-based program for homology modeling of proteins. Restraints are generated from the information contained in structures of homologous template proteins. Employing the restraints generated by PERMOL, three-dimensional structures are obtained using MD programs such as DYANA or CNS. In contrast to other programs PERMOL is mainly based on the use of dihedral angle information which is optimally suited to preserve the local secondary structure. The global arrangement of these elements is then facilitated by a small number of distance restraints. Using PERMOL homology, models of high quality are obtained. A key advantage of the proposed method is its flexibility, which allows the inclusion of data from other sources, such as experimental restraints and the use of modern molecular dynamics programs to calculate structures. AVAILABILITY: The software and a detailed manual are available free of charge (http://www.biologie.uni-regensburg.de/Biophysik/Kalbitzer/permol/permol.html)

Algorithms↗
PubMed2005

Conformational states of human H-Ras detected by high-field EPR, ENDOR, and 31P NMR spectroscopy.

Ras is a central constituent of the intracellular signal transduction that switches between its inactive state with GDP bound and its active state with GTP bound. A number of different X-ray structures are available. Different magnetic resonance techniques were used to characterise the conformational states of the protein and are summarised here. 31P NMR spectroscopy was used as probe for the environment of the phosphate groups of the bound nucleotide. It shows that in liquid solution additional conformational states in the GDP as well as in the GTP forms coexist which are not detected by X-ray crystallography. Some of them can also be detected by solid-state NMR in the micro crystalline state. EPR and ENDOR spectroscopy were used to probe the environment of the divalent metal ion (Mg2+ was replaced by Mn2+) bound to the nucleotide in the protein. Here again different states could be observed. Substitution of normal water by 17O-enriched water allowed the determination of the number of water molecules in the first coordination sphere of the metal ion. In liquid solution, they indicate again the existence of different conformational states. At low temperatures in the frozen state ENDOR spectroscopy suggests that only one state exists for the GDP- and GTP-bound form of Ras, respectively.

Electron Spin Resonance Spectroscopy↗
PubMed2005

Solution structure of human proinsulin C-peptide.

The C-peptide of proinsulin is important for the biosynthesis of insulin, but has been considered for a long time to be biologically inert. Recent studies in diabetic patients have stimulated a new debate about its possible regulatory role, suggesting that it is a hormonally active peptide. We describe structural studies of the C-peptide using 2D NMR spectroscopy. In aqueous solution, the NOE patterns and chemical shifts indicate that the ensemble is a nonrandom structure and contains substructures with defined local conformations. These are more clearly visible in 50% H2O/50% 2,2,2-trifluoroethanol. The N-terminal region (residues 2-5) forms a type I beta-turn, whereas the C-terminal region (residues 27-31) presents the most well-defined structure of the whole molecule including a type III'beta-turn. The C-terminal pentapeptide (EGSLQ) has been suggested to be responsible for chiral interactions with an as yet uncharacterized, probably a G-protein-coupled, receptor. The three central regions of the molecule (residues 9-12, 15-18 and 22-25) show tendencies to form beta-bends. We propose that the structure described here for the C-terminal pentapeptide is consistent with the previously postulated CA knuckle, believed to represent the active site of the C-peptide of human proinsulin.

Amino Acid Sequence↗
PubMed2004

Perturbation of the conformational equilibria in Ras by selective mutations as studied by 31P NMR spectroscopy.

Ras regulates a variety of different signal transduction pathways acting as molecular switch. It was shown by liquid and solid-state (31)P NMR spectroscopy that Ras exists in the guanosine-5'-(beta,gamma-imido)triphosphate bound form in at least two conformational states interconverting in millisecond time scale. The relative population between the two conformational states affects drastically the affinity of Ras to its effectors. (31)P NMR spectroscopy shows that the conformational equilibrium can be shifted specifically by point mutations, including mutations with oncogenic potential, thus modifying the effector interactions and their coupling to dynamic properties of the protein.

Amino Acid Sequence↗
PubMed2004

Solid-state 31P NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: comparison between crystalline and solution state.

Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.

Binding Sites↗
PubMed2004

Solution structures of the inactive and BeF3-activated response regulator CheY2.

The chemotactic signalling chain to the flagellar motor of Sinorhizobium meliloti features a new type of response regulator, CheY2. CheY2 activated by phosphorylation (CheY2-P) controls the rotary speed of the flagellar motor (instead of reversing the sense of rotation), and it is efficiently dephosphorylated by phospho-retrotransfer to the cognate kinase, CheA. Here, we report the NMR solution structures of the Mg(2+)-complex of inactive CheY2, and of activated CheY2-BeF(3), a stable analogue of CheY2-P, to an overall root mean square deviation of 0.042 nm and 0.027 nm, respectively. The 14 kDa CheY2 protein exhibits a characteristic open (alpha/beta)(5) conformation. Modification of CheY2 by BeF(3)(-) leads to large conformational changes of the protein, which are in the limits of error identical with those observed by phosphorylation of the active-centre residue Asp58. In BeF(3)-activated CheY2, the position of Thr88-OH favours the formation of a hydrogen bond with the active site, Asp58-BeF(3), similar to BeF(3)-activated CheY from Escherichia coli. In contrast to E.coli, this reorientation is not involved in a Tyr-Thr-coupling mechanism, that propagates the signal from the incoming phosphoryl group to the C-terminally located FliM-binding surface. Rather, a rearrangement of the Phe59 side-chain to interact with Ile86-Leu95-Val96 along with a displacement of alpha4 towards beta5 is stabilised in S.meliloti. The resulting, activation-induced, compact alpha4-beta5-alpha5 surface forms a unique binding domain suited for specific interaction with and signalling to a rotary motor that requires a gradual speed control. We propose that these new features of response regulator activation, compared to other two-component systems, are the key for the observed unique phosphorylation, dephosphorylation and motor control mechanisms in S.meliloti.

Amino Acid Sequence↗
PubMed2004

Conformational states of the small G protein Arf-1 in complex with the guanine nucleotide exchange factor ARNO-Sec7.

Arf1 is a small G protein involved in vesicular trafficking, and although it is only distantly related to Ras, it adopts a similar three-dimensional structure. In the present work, we study Arf1 bound to GDP and GTP and its interactions with one of its guanosine nucleotide exchange factors, ARNO-Sec7. The (31)P NMR spectra of Arf1.GDP.Mg(2+) and Arf1.GTP.Mg(2+) share the general features typical for all small G proteins studied so far. Especially, the beta-phosphate resonances of the bound nucleotide are shifted strongly downfield compared with the resonance positions of the free magnesium complexes of GDP and GTP. However, no evidence for an equilibrium between two conformational states of Arf1.GDP.Mg(2+) or Arf1.GTP.Mg(2+) could be observed as it was described earlier for Ras and Ran. Glu(156) of ARNO-Sec7 has been suggested to play as "glutamic acid finger" an important role in the nucleotide exchange mechanism. In the millimolar concentration range used in the NMR experiments, wild type ARNO-Sec7 and ARNO-Sec7(E156D) do weakly interact with Arf1.GDP.Mg(2+) but do not form a strong complex with magnesium-free Arf1.GDP. Only wild type ARNO-Sec7 competes weakly with GDP on Arf1.GDP.Mg(2+) and leads to a release of GDP when added to the solution. The catalytically inactive mutants ARNO-Sec7(E156A) and ARNO-Sec7(E156K) induce a release of magnesium from Arf1.GDP.Mg(2+) but do not promote GDP release. In addition, ARNO-Sec7 does not interact or only very weakly interacts with the GTP-bound form of Arf1, opposite to the observation made earlier for Ran, where the nucleotide exchange factor RCC1 forms a complex with Ran.GTP.Mg(2+) and is able to displace the bound GTP.

ADP-Ribosylation Factor 1↗