Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation

The force-dependent interaction between talin and vinculin plays a crucial role in the initiation and growth of focal adhesions. Here we use magnetic tweezers to characterise the mechano-sensitive compact N-terminal region of the talin rod, and show that the three helical bundles R1-R3 in this region unfold in three distinct steps consistent with the domains unfolding independently. Mechanical stretching of talin R1-R3 enhances its binding to vinculin and vinculin binding inhibits talin refolding after force is released. Mutations that stabilize R3 identify it as the initial mechano-sensing domain in talin, unfolding at ~5 pN, suggesting that 5 pN is the force threshold for vinculin binding and adhesion progression

Quantitative Evaluation of Cross-Peak Volumes in Multidimensional Spectra by Nonlinear-Least-Squares Curve Fitting

A procedure for quantitative evaluation of cross-peak volumes in spectra of any order of dimensions is described; this is based on a generalized algorithm for combining appropriate one-dimensional integrals obtained by nonlinear-least-squares curve-fitting techniques. This procedure is embodied in a program, NDVOL, which has three modes of operation: a fully automatic mode, a manual mode for interactive selection of fitting parameters, and a fast reintegration mode. The procedures used in the NDVOL program to obtain accurate volumes for overlapping cross peaks are illustrated using various simulated overlapping cross-peak patterns. The precision and accuracy of the estimates of cross-peak volumes obtained by application of the program to these simulated cross peaks and to a back-calculated 2D NOESY spectrum of dihydrofolate reductase are presented. Examples are shown of the use of the program with real 2D and 3D data. It is shown that the program is able to provide excellent estimates of volume even for seriously overlapping cross peaks with minimal intervention by the user. © 1995 Academic Press. All rights reserved.link_to_subscribed_fulltex

Direct measurement of the pK(a) of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: Implications for the catalytic mechanism

The ionization stale of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [C-13 gamma] aspartic acid and measuring the C-13 chemical shifts in the ape, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pK(a) greater than similar to 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pK(a) value of less than 4 in this complex. Such a low pK(a) value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pK(a) = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 C-13 gamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process

The conformation of coenzyme a bound to chloramphenicol acetyltransferase determined by transferred NOE experiments

The conformation of coenzyme A bound to chloramphenicol acetyltransferase has been studied in solution by NMR methods. Transferred nuclear Overhauser enhancement (NOE) and rotating frame NOE (ROE) experiments were used to determine the conformation of the bound coenzyme. Experiments were carried out at five mixing times and two temperatures, and with normal and perdeuterated enzyme, to ensure (1) that the fast exchange condition was satisfied and (2) that the results were not complicated by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation matrices. For the binary complex of coenzyme A (CoA) and enzyme, the conformation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the structure deduced by NMR is very close to that seen in the crystal structure of this complex, while the pantetheine moiety is clearly less extended. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular energy terms). The difference between the NMR and X-ray structures is interpreted in terms of the existence of two conformations of the CoA-enzyme complex. Support for this model comes from measurements of the coenzyme dissociation rate constant; NMR (lineshape analysis and transferred NOE experiments) gives estimates of k(off) ~ 3700 s-1 at 298 K and ~ 500 s-1 at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the ternary complex of CoA, chloramphenicol and enzyme, 71 NOE cross-peaks between protons of coenzyme A and a further ten cross-peaks between protons of coenzyme A and chloramphenicol were measured. Starting with a model derived from the crystal structures of the two binary complexes (in the absence of crystallographic data for the ternary complex) the conformations and relative positions of the two ligands were refined using the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, while the pantetheine arm is more extended and approaches close to the bound chloramphenicol molecule. The model of the ternary complex is discussed in terms of the information available on the mechanism of the enzyme.link_to_subscribed_fulltex