Toward universal protein post-translational modification detection in high throughput format.

Post-translational modification (PTM) of proteins plays essential regulatory roles in a variety of pathological conditions. Reliable and practical assays are required to accelerate the discovery of inhibitors and activators for PTM related diseases. Today, methodologies are based on specific or group-specific PTM recognition of e.

Comparative analyses of the thermodynamic RNA binding signatures of different types of RNA recognition motifs.

RNA recognition motifs (RRMs) are structurally versatile domains important in regulation of alternative splicing. Structural mechanisms of sequence-specific recognition of single-stranded RNAs (ssRNAs) by RRMs are well understood. The thermodynamic strategies are however unclear.

Real-time assessment of possible electromagnetic-field-induced changes in protein conformation and thermal stability.

Previous studies on possible interactions of radiofrequency electromagnetic fields (RF EMFs) with proteins have suggested that RF EMFs might affect protein structure and folding kinetics. In this study, the isolated thermosensor protein GrpE of the Hsp70 chaperone system of Escherichia coli was exposed to EMFs of various frequencies and field strengths under strictly controlled conditions. Circular dichroism spectroscopy was used to monitor possible structural changes.

The RING domain of the scaffold protein Ste5 adopts a molten globular character with high thermal and chemical stability.

Ste5 is a scaffold protein that controls the pheromone response of the MAP-kinase cascade in yeast cells. Upon pheromone stimulation, Ste5 (through its RING-H2 domain) interacts with the β and γ subunits of an activated heterodimeric G protein and promotes activation of the MAP-kinase cascade. With structural and biophysical studies, we show that the Ste5 RING-H2 domain exists as a molten globule under native buffer conditions, in yeast extracts, and even in denaturing conditions containing urea (7 M).

Experimental system for real-time assessment of potential changes in protein conformation induced by electromagnetic fields.

A novel experimental system to distinguish between potential thermal and non-thermal effects of electromagnetic fields (EMFs) on the conformational equilibrium and folding kinetics of proteins is presented. The system comprises an exposure chamber installed within the measurement compartment of a spectropolarimeter and allows real-time observation of the circular dichroism (CD) signal of the protein during EMF exposure. An optical temperature probe monitors the temperature of the protein solution at the site of irradiation.

The high kinetic stability of a G-quadruplex limits hnRNP F qRRM3 binding to G-tract RNA.

The RNA binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in telomeres maintenance and pre-mRNA processing, such as alternative splicing and polyadenylation. It specifically recognizes RNA containing three consecutive guanines (G-tracts) that have the potential to assemble into G-quadruplexes. We have proposed recently that hnRNP F could regulate alternative splicing by remodeling RNA structures, such as G-quadruplexes.

Probing the energetic and kinetic impact of topologically conserved interactions in the SIV gp41 six-helix bundle.

In this study we used an engineered six-helix bundle construct corresponding to the fusogenic core of the SIV gp41 protein as a model system to investigate the folding of a trimeric protein, which acquires a compact structure upon association of largely unstructured monomeric peptides. Thirteen mutants were generated in order to gain information about the thermodynamic and kinetic roles of topologically conserved tertiary interactions to folding and stability. The effect of the mutations was assessed by circular dichroism spectroscopy from urea-induced equilibrium unfolding experiments and in time-resolved mode to follow the kinetics of refolding and unfolding.

Ligand binding studied by 2D IR spectroscopy using the azidohomoalanine label.

We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coefficient of 350-400 M(-1) cm(-1) is high enough to routinely measure it in the low millimolar concentration regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain.

Her2-specific multivalent adapters confer designed tropism to adenovirus for gene targeting.

Adenoviruses (Ads) hold great promise as gene vectors for diagnostic or therapeutic applications. The native tropism of Ads must be modified to achieve disease site-specific gene delivery by Ad vectors and this should be done in a programmable way and with technology that can realistically be scaled up. To this end, we applied the technologies of designed ankyrin repeat proteins (DARPins) and ribosome display to develop a DARPin that binds the knob domain of the Ad fiber protein with low nanomolar affinity (K(D) 1.

Survey of the year 2008: applications of isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is a fast, accurate and label-free method for measuring the thermodynamics and binding affinities of molecular associations in solution. Because the method will measure any reaction that results in a heat change, it is applicable to many different fields of research from biomolecular science, to drug design and materials engineering, and can be used to measure binding events between essentially any type of biological or chemical ligand. ITC is the only method that can directly measure binding energetics including Gibbs free energy, enthalpy, entropy and heat capacity changes.

Disabling c-Myc in childhood medulloblastoma and atypical teratoid/rhabdoid tumor cells by the potent G-quadruplex interactive agent S2T1-6OTD.

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex-forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes.

Molecular insights into mammalian end-binding protein heterodimerization.

Microtubule plus-end tracking proteins (+TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of +TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse +TIPs to growing microtubule plus ends.

A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase.

YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis, reduces quinones via a ping-pong bi-bi mechanism with a pronounced preference for NADPH. The enzyme occurs as a stable tetramer in solution. The two extended dimer surfaces are packed against each other by a 90 rotation of one dimer with respect to the other.

Defining the role of salt bridges in protein stability.

Although the energetic balance of forces stabilizing proteins has been established qualitatively over the last decades, quantification of the energetic contribution of particular interactions still poses serious problems. The reasons are the strong cooperativity and the interdependence ofnoncovalent interactions. Salt bridges are a typical example.

A survey of the year 2007 literature on applications of isothermal titration calorimetry.

Elucidation of the energetic principles of binding affinity and specificity is a central task in many branches of current sciences: biology, medicine, pharmacology, chemistry, material sciences, etc. In biomedical research, integral approaches combining structural information with in-solution biophysical data have proved to be a powerful way toward understanding the physical basis of vital cellular phenomena. Isothermal titration calorimetry (ITC) is a valuable experimental tool facilitating quantification of the thermodynamic parameters that characterize recognition processes involving biomacromolecules.

Kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart.

An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (-TDeltaDeltaS=5.

A survey of the year 2006 literature on applications of isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is a fast and robust method to determine the energetics of association reactions in solution. The changes in enthalpy, entropy and heat capacity that accompany binding provide unique insights into the balance of forces driving association of molecular entities. ITC is used nowadays on a day-to-day basis in hundreds of laboratories.

Thermodynamics of b-HLH-LZ protein binding to DNA: the energetic importance of protein-DNA contacts in site-specific E-box recognition by the complete gene product of the Max p21 transcription factor.

The Myc/Mad/Max network of dimeric basic region-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factors bind to enhancer box sequences (E-box) in the promotors of a large set of genes that control cell metabolism, proliferation, and differentiation. Max (Myc-associated factor X) is the obligate heterodimerization partner of Myc and Mad proteins. On the other hand, Max is the only member of the family capable of forming a stable homodimer.

Structure-function relationship of CAP-Gly domains.

In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of p150(glued) and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly-mediated protein interactions.

Electrostatic contribution to the thermodynamic and kinetic stability of the homotrimeric coiled coil Lpp-56: A computational study.

The protein moiety of the Braun's E. coli outer membrane lipoprotein (Lpp-56) is an attractive object of biophysical investigation in several aspects. It is a homotrimeric, parallel coiled coil, a class of coiled coils whose stability and folding have been studied only occasionally.

Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA.

Molecular basis of coiled-coil formation.

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed "trigger sequence," that is indispensable for folding.

Energetics of peptide recognition by the second PDZ domain of human protein tyrosine phosphatase 1E.

Formation of protein-protein assemblies is essential in maintaining cell structure and function. Conservation of structural motifs and binding sites is the result of evolutionary pressure for solutions compatible with both molecular economy and regulation. PDZ domains are a typical example: A conserved fold governs specificity toward recognition of C-terminal protein sequences by small sequential and/or structural deviations within a canonical binding mode.