Bio: Karin Welfle is an academic researcher from Max Delbrück Center for Molecular Medicine. The author has contributed to research in topics: Circular dichroism & Peptide. The author has an hindex of 14, co-authored 22 publications receiving 675 citations.
TL;DR: Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism, fluorescence, and differential scanning calorimetry (DSC) measurements.
Abstract: Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively).
TL;DR: In vivo studies reveal a short halflife of the antitoxin and a long lifetime of the ζ toxin and when transcriptiontranslation of a plasmid containing the and ζ genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the protein from the background.
Abstract: Streptococcus pyogenes pSM19035-encoded epsilon (10.7 kDa) and zeta (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with zeta acting as toxin that kills plasmid-deprived cells and epsilon as an antitoxin that neutralises the activity of zeta. The epsilon and zeta proteins co-purify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as epsilon2zeta2 heterotetramer in solution. Co-crystals of the epsilon2zeta2 complex contain epsilon and zeta in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the zeta protein has a significantly lower thermodynamic stability than the epsilon protein both in free state and in the complex. Proteolytic studies indicate that zeta protein is more stable in the epsilon2zeta2 complex than in the free state. In vivo studies reveal a short half-life of the epsilon antitoxin (-18 min) and a long lifetime of the zeta toxin (>60 min). When transcription-translation of a plasmid containing the epsilon and zeta genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the epsilon protein from the background.
TL;DR: The results indicate that IF2 C-2 is a globular molecule containing predominantly β structures (25% antiparallel and 8% parallel β strands and turns) and turns whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF1 C-1.
Abstract: Previous protein unfolding studies had suggested that IF2 C, the 24.5-kDa fMet-tRNA binding domain of Bacillus stearothermophilus translation initiation factor IF2, may consist of two subdomains. In the present work, the four Phe residues of IF2 C (positions 531, 599, 657, and 721) were replaced with Trp, yielding four variant proteins having intrinsic fluorescence markers in different positions of the molecule. Comparison of the circular dichroism and Trp fluorescence changes induced by increasing concentrations of guanidine hydrochloride demonstrated that IF2 C indeed consists of two subdomains: the more stable N-terminal (IF2 C-1) subdomain containing Trp-599, and the less stable C-terminal (IF2 C-2) subdomain containing Trp-721. Isolated subdomain IF2 C-2, which consists of just 110 amino acids (from Glu-632 to Ala-741), was found to bind fMet-tRNA with the same specificity and affinity as native IF2 or IF2 C-domain. Trimming IF2 C-2 from both N and C termini demonstrated that the minimal fragment still capable of fMet-binding consists of 90 amino acids. IF2 C-2 was further characterized by circular dichroism; by urea-, guanidine hydrochloride-, and temperature-induced unfolding; and by differential scanning calorimetry. The results indicate that IF2 C-2 is a globular molecule containing predominantly β structures (25% antiparallel and 8% parallel β strands) and turns (19%) whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF2 C-1.
TL;DR: Differences in thermodynamic properties between peptide-complexed HLA-B27 subtypes are correlated with a variety of structural properties, and the crystal structure of the B*2709 subtype complexed with a decameric peptide is presented.
Abstract: Selected HLA-B27 subtypes are associated with spondyloarthropathies, but the underlying mechanism is not understood. To explain this association in molecular terms, a comparison of peptide-dependent dynamic and structural properties of the differentially disease-associated subtypes HLA-B*2705 and HLA-B*2709 was carried out. These molecules differ only by a single amino acid at the floor of the peptide binding groove. The thermostabilities of a series of HLA-B27 molecules complexed with nonameric and decameric peptides were determined and revealed substantial differences depending on the subtype as well as the residues at the termini of the peptides. In addition we present the crystal structure of the B*2709 subtype complexed with a decameric peptide. This structure provides an explanation for the preference of HLA-B27 for a peptide with an N-terminal arginine as secondary anchor and the lack of preference for tyrosine as peptide C terminus in B*2709. The data show that differences in thermodynamic properties between peptide-complexed HLA-B27 subtypes are correlated with a variety of structural properties.
TL;DR: Data suggest that each heptad defines an operator half-site and that tight binding of the symmetric omega2 to the central 5'-TCA-3' core of symmetric or asymmetric targets (differently oriented heptads) is probably achieved by structural changes of DNA and/or protein or both.
Abstract: pSM19035-encoded ω protein forms a dimer (ω2) that binds to a set of 7-bp repeats with sequence 5′-NATCACN-3′. Upon binding to its cognate sites, ω2 regulates transcription of genes required for copy number control and stable inheritance of plasmids, and promotes accurate plasmid segregation. Protein ω2 binds poorly to one heptad but the affinity to DNA increases with two and more unspaced heptads in direct or inverted orientation. DNA titration of increasing numbers of heptads with ω2, monitored by circular dichroism measurements, indicates the binding of one ω2 to one heptad (ω2:heptad stoichiometry of 1:1). Spacing of two directly or inversely oriented heptads by 1 to 7 bp reduces the affinity of the protein for its cognate target site. The binding affinity of ω2 for two directly repeated heptads was severely reduced if one of the base pairs of the core 5′-ATCAC-3′ sequence of one of the heptads was individually substituted by any other base pair. Hydroxyl radical footprinting shows a protection pattern at the 5′-ATCAC-3′ core. These data suggest that each heptad defines an operator half-site and that tight binding of the symmetric ω2 to the central 5′-TCA-3′ core of symmetric or asymmetric targets (differently oriented heptads) is probably achieved by structural changes of DNA and/or protein or both.
TL;DR: The structure and function of antibodies and the mechanisms of physical and chemical instabilities are reviewed and various aspects of formulation development have been examined to identify the critical attributes for the stabilization of antibodies.
Abstract: The number of therapeutic monoclonal antibody in development has increased tremendously over the last several years and this trend continues. At present there are more than 23 approved antibodies on the US market and an estimated 200 or more are in development. Although antibodies share certain structural similarities, development of commercially viable antibody pharmaceuticals has not been straightforward because of their unique and somewhat unpredictable solution behavior. This article reviews the structure and function of antibodies and the mechanisms of physical and chemical instabilities. Various aspects of formulation development have been examined to identify the critical attributes for the stabilization of antibodies.
TL;DR: This chapter summarizes the experimental information on protein energetics and investigates the correlation between thermodynamic and structural characteristics of protein, including the water-ASA of various groups in the native and unfolded states, the number of hydrogen bonds, and the extent of van der Waals contacts in thenative state.
Abstract: Publisher Summary This chapter summarizes the experimental information on protein energetics. This field is developing fast and the concept of the energetics of protein structure has changed considerably during the past few years based on new findings. The proteins which are presented in the chapter are selected from a large number of proteins for which the thermodynamics of unfolding are studied in laboratory. The analysis of protein energetics presented in this chapter is based on several assumptions: (1) protein groups contribute additively, and proportionally as their surfaces, to the overall thermodynamic effects of unfolding; (2) the protein interior closely resembles an organic crystal in the way groups are packed and the energetics of the interactions among these groups are similar to those in the organic crystals; and (3) under certain conditions the denatured protein can be regarded as unfolded. The main criteria in choosing these proteins have been the reversibility of the denaturation process modeling unfolding, the completeness of this unfolding, the reliability of thermodynamic data on this process, and the resolution of the three dimensional structure of the given protein. The latter is important to investigate the correlation between thermodynamic and structural characteristics of protein, including the water-ASA of various groups in the native and unfolded states, the number of hydrogen bonds, and the extent of van der Waals contacts in the native state.
TL;DR: Evidence now indicates that toxin–antitoxin loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.
Abstract: Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.
TL;DR: All the fragments are rather resistant to heat‐induced denaturation, and display high conformational stabilities, which has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered.
Abstract: A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, β-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N⇋U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent Tm = 60–80°C), and display high conformational stabilities (ΔG(H2O) = 30–60 kJ mole−1). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications.
TL;DR: The dissection of the interaction of the toxins with intracellular targets and the elucidation of the tertiary structures of toxin-antitoxin complexes have provided exciting insights into toxin-antsitoxin behavior.
Abstract: Antibiotic resistance, virulence, and other plasmids in bacteria use toxin-antitoxin gene pairs to ensure their persistence during host replication. The toxin-antitoxin system eliminates plasmid-free cells that emerge as a result of segregation or replication defects and contributes to intra- and interspecies plasmid dissemination. Chromosomal homologs of toxin-antitoxin genes are widely distributed in pathogenic and other bacteria and induce reversible cell cycle arrest or programmed cell death in response to starvation or other adverse conditions. The dissection of the interaction of the toxins with intracellular targets and the elucidation of the tertiary structures of toxin-antitoxin complexes have provided exciting insights into toxin-antitoxin behavior.