Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.

We develop a general minimally coupled subspace approach (MCSA) to compute absolute entropies of macromolecules, such as proteins, from computer generated canonical ensembles. Our approach overcomes limitations of current estimates such as the quasi-harmonic approximation which neglects non-linear and higher-order correlations as well as multi-minima characteristics of protein energy landscapes. Here, Full Correlation Analysis, adaptive kernel density estimation, and mutual information expansions are combined and high accuracy is demonstrated for a number of test systems ranging from alkanes to a 14 residue peptide. We further computed the configurational entropy for the full 67-residue cofactor of the TATA box binding protein illustrating that MCSA yields improved results also for large macromolecular systems.

Use the force: Force–volume atomic force microscopy (AFM) can image native membrane proteins and quantify and map their chemical and physical properties at molecular resolution (see images). For the light‐driven proton pump bacteriorhodopsin (BR), the data shows that lipids form a flexible framework embedding a mechanically anisotropic proton pump, and that the BR adopts different structurally stable conformations that are important for proton pumping.

Proteins are usually described and classified according to amino acid sequence, structure or function. Here, we develop a minimally biased scheme to compare and classify proteins according to their internal mobility patterns. This approach is based on the notion that proteins not only fold into recurring structural motifs but might also be carrying out only a limited set of recurring mobility motifs. The complete set of these patterns, which we tentatively call the dynasome, spans a multi-dimensional space with axes, the dynasome descriptors, characterizing different aspects of protein dynamics. The unique dynamic fingerprint of each protein is represented as a vector in the dynasome space. The difference between any two vectors, consequently, gives a reliable measure of the difference between the corresponding protein dynamics. We characterize the properties of the dynasome by comparing the dynamics fingerprints obtained from molecular dynamics simulations of 112 proteins but our approach is, in principle, not restricted to any specific source of data of protein dynamics. We conclude that: 1. the dynasome consists of a continuum of proteins, rather than well separated classes. 2. For the majority of proteins we observe strong correlations between structure and dynamics. 3. Proteins with similar function carry out similar dynamics, which suggests a new method to improve protein function annotation based on protein dynamics.

Cyclic nucleotide-regulated ion channels are present in bacteria, plants, vertebrates, and humans. In higher organisms, they are closely involved in signaling networks of vision and olfaction. Binding of cAMP or cGMP favors the activation of these ion channels. Despite a wealth of structural and studies, there is a lack of structural data describing the gating process in a full-length cyclic nucleotideregulated channel. We used high-resolution atomic force microscopy (AFM) to directly observe the conformational change of the membrane embedded bacterial cyclic nucleotide-regulated channel MlotiK1. In the nucleotide-bound conformation, the cytoplasmic cyclic nucleotide-binding (CNB) domains of MlotiK1 are disposed in a fourfold symmetric arrangement forming a pore-like vestibule. Upon nucleotide-unbinding, the four CNB domains undergo a large rearrangement, stand up by ∼1.7 nm, and adopt a structurally variable grouped conformation that closes the cytoplasmic vestibule. This fully reversible conformational change provides insight into how CNB domains rearrange when regulating the potassium channel.conformational changes | cyclic nucleotide gating | membrane protein | MloK1 | single-molecule imaging P otassium channels are tetrameric membrane proteins that facilitate the permeation of potassium ions through the membrane with high specificity and high-throughput rates. These channels are central to the electrical activity of cells in humans and are, therefore, of fundamental importance for the function of nervous and muscular systems. The major mode of functional regulation in potassium channels is gating, a conformational change that occurs on the intracellular regions of the ion pore domain and involves an iris-like movement of the C-terminal transmembrane helices and a widening of the intracellular pore. Gating in potassium channels is induced by a variety of stimuli, including membrane voltage, intracellular calcium concentration, and cyclic nucleotide levels (1). These stimuli are sensed by a separate domain from the ion pore domain, inducing a conformational change that is then propagated to the gate of the channel.The MlotiK1 potassium channel, from the bacterium Mesorhizobium loti, belongs to the family of channels that is regulated by cyclic nucleotides and includes eukaryotic cyclic nucleotide-gated (CNG) and hyperpolarization activated cyclic nucleotide-gated (HCN) channels (2, 3). These channels have C-terminal cytoplasmic cyclic nucleotide-binding (CNB) domains and upon binding of cAMP or cGMP, these domains undergo a conformational change that favors the opening of the gate of the channel. The major difference between the MlotiK1 channel and the CNG or HCN channels is the linker that connects the gate to the CNB domains. This helical linker (C linker) is roughly 80 residues long in CNG and HCN channels and only ∼20 residues long in the MlotiK1 channel.The MlotiK1 channel has been the focus of structural and functional studies with the aim of understanding channel regulation by cyclic nucleotides. X-ray...

Insulin aggregation critically depends on pH. The underlying energetic and structural determinants are, however, unknown. Here, we measure the kinetics of the primary aggregation steps of the insulin monomer in vitro and relate it to its conformational flexibility. To assess these primary steps the monomer concentration was monitored by mass spectrometry at various pH values and aggregation products were imaged by atomic force microscopy. Lowering the pH from 3 to 1.6 markedly accelerated the observed aggregation kinetics. The influence of pH on the monomer structure and dynamics in solution was studied by molecular dynamics simulations, with the protonation states of the titrable groups obtained from electrostatic calculations. Reduced flexibility was observed for low pH values, mainly in the C terminus and in the helix of the B chain; these corresponded to an estimated entropy loss of 150 J mol(-1) K(-1). The striking correlation between entropy loss and pH value is consistent with the observed kinetic traces. In analogy to the well-known Phi value analysis, this result allows the extraction of structural information about the rate determining transition state of the primary aggregation steps. In particular, we suggest that the residues in the helix of the B chain are involved in this transition state.

A method is presented to evaluate a molecule's entropy from the atomic forces calculated in a molecular dynamics simulation. Specifically, diagonalization of the mass-weighted force covariance matrix produces eigenvalues which in the harmonic approximation can be related to vibrational frequencies. The harmonic oscillator entropies of each vibrational mode may be summed to give the total entropy. The results for a series of hydrocarbons, dialanine and a β hairpin are found to agree much better with values derived from thermodynamic integration than results calculated using quasiharmonic analysis. Forces are found to follow a harmonic distribution more closely than coordinate displacements and better capture the underlying potential energy surface. The method's accuracy, simplicity, and computational similarity to quasiharmonic analysis, requiring as input force trajectories instead of coordinate trajectories, makes it readily applicable to a wide range of problems.

The quasiharmonic approximation is the most widely used estimate for the configurational entropy of macromolecules from configurational ensembles generated from atomistic simulations. This method, however, rests on two assumptions that severely limit its applicability, (i) that a principal component analysis yields sufficiently uncorrelated modes and (ii) that configurational densities can be well approximated by Gaussian functions. In this paper we introduce a nonparametric density estimation method which rests on adaptive anisotropic kernels. It is shown that this method provides accurate configurational entropies for up to 45 dimensions thus improving on the quasiharmonic approximation. When embedded in the minimally coupled subspace framework, large macromolecules of biological interest become accessible, as demonstrated for the 67-residue coldshock protein.