Changement conformationnel | EPFL Graph Search

vignette|Exemple de changement conformationnel En biochimie, un changement conformationnel est la transition entre deux géométries moléculaires, souvent induite par des facteurs environnementaux. Une macromolécule est le plus souvent flexible et dynamique, chaque configuration tridimensionnelle possible définissant une conformation. La forme d'une macromolécule peut se modifier en réponse à un changement dans les paramètres environnementaux tels que la température, le pH (acidité, basicité), le champ électrique, la salinité, la lumière (sur les chromophores), la phosphorylation ou la liaison d'un ligand. Ces modifications structurelles peuvent être d'ampleur variable, de l'ordre de quelques dizaines de picomètres jusqu'au nanomètre, et de rapidité également très variable, de l'ordre de la nanoseconde jusqu'à la seconde ; elles sont associées à des phénomènes physiologiques tels que l'allostérie et la catalyse des enzymes. Les changements conformationnels jouent un rôle fonctionnel im

Large-scale filament formation inhibits the activity of CTP synthetase

Anne-Florence Raphaëlle Bitbol, Hsin-Jung Li

Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

eLife Sciences Publications, Ltd2014

Structural and functional analysis of host attachment proteins from contractile injection systems

Michel Plattner

Bacteriophages of the myoviridae family, R-type pyocin, Photorhabus virulence cassette (PVC), Serratia antifeeding prophage (Afp) and type 6 secretion system (T6SS) form a class of contractile injection systems that share common structural and functional components. The baseplate is a complex macromolecular structure that can change conformation. The tail is a rigid tube surrounded by a contractile sheath. They efficiently translocate genetic material (bacteriophages), deliver toxins into the external milieu or neighboring cells (PVC, Afp and T6SS) and efficiently kill competitor bacteria by depolarizing the membrane potential (Pyocin). The host range of bacteriophages or target spectrum of R-type pyocins, PVC, Afps is determined by filamentous tail fibers and more bulky tailspikes (TSP) that emanate from the baseplate. The recently discovered bacteriophage CBA120 encodes six putative host attachment proteins, of which four (named TSP1- TSP4) show some sequence similarity to known tailspikes. Despite four different proteins, CBA120 was found to be highly specific toward one host, namely E. coli O157:H7, a frequent contaminant of ground beef and a well-known human pathogen. In this work the crystallographic structure of TSP2, TSP3 and TSP4 are presented. In addition, functional assays suggest that CBA120 has at least three potential hosts. The structure of TSP1 was solved elsewhere but no activity was found towards E. coli O157:H7 or any other host. To better understand how the four TSP bind the phage particle, the TSP complex was formed in vitro and a new complex formation pathway was suggested. In addition to the CBA120, crystal structures of two TSP from Acinetobacter baumannii specific phages were solved. Acinetobacter baumannii is a highly drug resistant âflesh-eatingâ bacterium. Studies conducted with R-type pyocin show that its host specificity can be manipulated by changing the host attachment fiber. It was also shown that the native fiber protein could be exchanged for TSP proteins. In order to be incorporated in the pyocin particle, a new TSP can be fused to a 164 residue long N-terminal domain of the native fiber. This fragment is critical for maintaining the integrity of the baseplate and for transmitting the target surface binding signal to it. To obtain the structure of this fragment, we created a fusion of this domain with a well-crystallizable TSP. Several other proteins of the pyocin baseplate were cloned, expressed and purified. The result of these experiments was the determination of the crystal structure of the proteolytically stable fragment of PA0618, the largest protein of the pyocin baseplate. PA0618 is orthologous to T4 gp6, which is responsible of circularization of the baseplate. Taken together the structures of five previously unknown TSP and the proteolytically stable fragment of PA0618 were determined. The acquired structural information will help to better understand the relationship of peptide sequence, structure and function, in particular, the interaction of TSP and fiber proteins with polysaccharides that decorate the bacterial surface. Furthermore, all crystal structures reported here were determined by de novo phasing, and this structural information will further improve the ability to predict protein structure. Thanks to the enzymatic activity of the TSP proteins and their ability to specifically target bacterial cells, these proteins can be used in antimicrobial applications.

EPFL2018

Chemical exchange in proteins

Mariachiara Verde

Nuclear Magnetic Resonance (NMR) spectroscopy offers many ways to investigate dynamic properties of molecules. A wide variety of experimental techniques can probe molecular dynamics on time scales that range from 10-12 to 103 seconds. Many lines of evidence point to the existence of large scale dynamics on milli- to microsecond time scale that are essential to molecular function. Those conformational motions can give rise to chemical exchange effects. So far in the literature two methods have been mainly used to study such processes: CPMG echo trains and R1ρ spin-lock relaxation experiments. The former is suited to investigate processes on the millisecond time scale, whereas R1ρ can probe processes ranging from milliseconds down to ca. microseconds. CPMG and R1ρ studies of the relaxation rates of single quantum (SQ) coherences can be complemented by multiple quantum coherence (MQ) coherences experiments which can provide information whether two (or more) spins are affected simultaneously by chemical exchange. The goal of this thesis is to characterize chemical exchange in proteins by new (Heteronuclear Double Resonance, HDR) and existing (relaxation compensated CPMG) methods. The presence of chemical exchange has been identified in APO-rMUP using the classical CPMG experiment on single quantum 15N magnetization, whereas in HOLO-rMUP no presence of chemical exchange has been found. Most of the thesis is dedicated to the analysis and the application of a new method, HDR, which is designed to determine the cross-relaxation rates between MQ operators. In fact, in heteronuclear systems, correlated chemical exchange contributes to cross-relaxation between MQ coherences 2IxSx and 2IySy. This method, based on MLEV-32 and WALTZ-16 used in double resonance mode, is designed to effectively preserve all relevant MQ coherences simultaneously so that the interconversion between MQ operators can only arise through cross-relaxation. This is an essential requirement if we want to measure, for example, small cross-relaxation rates between MQ operators. We have treated the effects of coherent evolution during the HDR sequences by numerical simulations and Average Hamiltonian Theory (AHT) showing that, under most conditions, the dynamics of MQ coherences is not affected by coherent evolution. This result is proved experimentally on the 15N-1H pair of selectively deuterated tBoc-glycine. The effect of relaxation during HDR sequences leads to an effective relaxation superoperator which is explored by numerical simulations and predicted by Average Liouvillian Theory (ALT). Finally the HDR MLEV-32 sequence is applied to amide 1H-15N pairs in ubiquitin and KIX proteins. In the case of ubiquitin the HDR MLEV-32 experiments and the numerical simulations run with parameters obtained from the literature are in good agreement, suggesting that HDR sequences could be used as a tool to obtain information about the dynamics of the system.

EPFL2009