Biomedical subjects

Ulrich L Günther

Publications and source records attributed to Ulrich L Günther.

7 recordsLinked to original sources

PubMed2006

Retinol modulates site-specific mobility of apo-cellular retinol-binding protein to promote ligand binding.

A fundamental question in protein science is how the inherent dynamics of a protein influence its function. If this function involves interactions with a ligand, the protein-ligand encounter has the potential to modulate the protein dynamics. This study reveals how site-specific mobility can be modulated by the ligand to facilitate high affinity binding. We have investigated the mechanism of retinol uptake by the cellular retinol-binding protein type I (CRBP) using line shape analysis of NMR signals. The highly similar structures of apo- and holo-CRBP exhibit closed conformations that seemingly offer no access to ligand, yet the protein binds retinol rapidly and with high affinity. NMR line shape analysis reveals how protein dynamics resolve this apparent paradox. An initial nonspecific encounter with the ligand induces the formation of long-lived conformers in the portal region of CRBP suggesting a mechanism how retinol accesses the cavity.

Binding Sites↗

PubMed2005

De novo design of a stable N-terminal helical foldamer.

A peptide NTH-18 was synthesized in which a N-terminal helix is stabilised by two crossed disulfide bonds to a C-terminal extension. The design was inspired by the structure of the neurotoxic peptide apamin, which has previously been used to stabilise helices in miniature enzymes. CD- and NMR-spectroscopy indicated that NTH-18 adopted a fold similar to that found in apamin. However, the arrangement of the elements of secondary structures was inverted relative to apamin; a N-terminal alpha-helix was connected by a reverse turn to a C-terminal extension of non-canonical secondary structure. NTH-18 displayed significant stability to heat and changes of pH. The high definition of the N-terminal end of the alpha-helix of NTH-18 should make this peptide a useful vehicle to stabilise alpha-helices in proteins with applications in protein engineering and molecular recognition.

Amino Acid Sequence↗

PubMed2004

Tracing kinetic intermediates during ligand binding.

Specific protein-ligand interactions are central to biological control. Although structure determination provides important insight into these interactions, it does not address dynamic events that occur during binding. While many biophysical techniques can provide a global view of these dynamics, NMR can be used to derive site-specific dynamics at atomic resolution. Here we show how NMR line shapes can be analyzed to identify long-lived kinetic intermediates for individual amino acids on the reaction pathway for a protein-ligand interaction. Different ligands cause different intermediate states. The lifetimes of these states determine the specificity of binding. This novel approach provides a direct, site-specific visualization of the kinetic mechanism of protein-ligand interactions.

Antigens, Polyomavirus Transforming↗

PubMed2003

Nuclear magnetic resonance structure of the P395S mutant of the N-SH2 domain of the p85 subunit of PI3 kinase: an SH2 domain with altered specificity.

Understanding the specificity of Src homology 2 (SH2) domains is important because of their critical role in cell signaling. Previous genetic analysis has characterized mutants of the N-terminal src homology 2 (SH2) domain of the p85 subunit of phosphoinositide 3-kinase (PI3K). The P395S mutant exhibits a specificity for phosphopeptide binding different from that of the wild-type SH2. The P395S mutant has an increased affinity for the platelet-derived growth factor receptor (PDGFr) compared to polyomavirus middle T antigen (MT). Solution structures of the P395S mutant of the p85 N-SH2 alone and complexed to a PDGFr phosphopeptide were determined to explain the change in specificity. Chemical shift perturbations caused by different peptides were compared for mutant and wild-type structures. The results show that the single P395S mutation has broad effects on the structure. Furthermore, they provide a rationale for the observed changes in binding preference.

Amino Acid Sequence↗

PubMed2003

Direct observation of protein-ligand interaction kinetics.

Internal dynamics on the micro- to millisecond time scale have a strong influence on the affinity and specificity with which a protein binds ligands. This time scale is accessible through relaxation dispersion measurements using NMR. By studying the dynamics of a protein with different concentrations of a ligand, one can determine the dynamic effects induced by the ligand. Here we have studied slow internal dynamics of the N-terminal src homology 2 domain of phosphatidylinositide 3-kinase to probe the role of individual residues for the interaction with a tyrosine-phosphorylated binding sequence from polyoma middle T antigen. While slow dynamic motion was restricted to a few residues in the free SH2 and in the SH2 complex, motion was significantly enhanced by adding even small amounts of ligand. Kinetic rates induced by ligand binding varied between 300 and 2000 s(-1). High rates reflected direct interactions with the ligand or rearrangements caused by ligand binding. Large differences in rates were observed for residues adjacent in the primary sequence reflecting their individual roles in ligand interaction. However, rates were similar for residues involved in the same side chain interactions, reflecting concerted motions during ligand binding. For a subset of residues, exchange must involve structural intermediates which play a crucial role in high-affinity ligand binding. This analysis supports a new view of the dynamics of individual sites of a protein during ligand interaction.

Amino Acid Sequence↗

PubMed2002

WAVEWAT-improved solvent suppression in NMR spectra employing wavelet transforms.

WAVEWAT is a new processing algorithm to suppress the on-resonance water signal in NMR spectra. It is based on a multiresolution analysis (MRA) of the free induction decay (FID) using a dyadic discrete wavelet transform (DWT). The width of the suppressed signal can be adjusted so that signals close to water are recovered without distortion of the signal shape and intensity. Computational efficiency is comparable to that of convolution filters employing a Fourier transform.

Algorithms↗

PubMed2002

NMRKIN: simulating line shapes from two-dimensional spectra of proteins upon ligand binding.

The analysis of the shape of signals in NMR spectra is a powerful tool to study exchange and reaction kinetics. Line shapes in two-dimensional spectra of proteins recorded for titrations with ligands provide information about binding rates observed at individual residues. Here we describe a fast method to simulate a series of line shapes derived from two-dimensional spectra of a protein during a ligand titration. This procedure, which takes the mutual effects of two dimensions into account, has been implemented in MATLAB as an add-on to NMRLab (Gunther et al., 2000). In addition, more complex kinetic models, including sequential and parallel reactions, were simulated to demonstrate common features of more complex line shapes which could be encountered in protein-ligand interactions. As an example of this method, we describe its application to line shapes obtained for a titration of the p85 N-SH2 domain of PI3-kinase with a peptide derived from polyomavirus middle T antigen (MT).

Algorithms↗