Determination of Conformational Equilibria in Proteins Using Residual Dipolar Couplings

In order to carry out their functions, proteins often undergo significant conformational fluctuations that enable them to interact with their partners. The accurate characterization of these motions is key in order to understand the mechanisms by which macromolecular recognition events take place. Nuclear magnetic resonance spectroscopy offers a variety of powerful methods to achieve this result. We discuss a method of using residual dipolar couplings as replica-averaged restraints in molecular dynamics simulations to determine large amplitude motions of proteins, including those involved in the conformational equilibria that are established through interconversions between different states. By applying this method to ribonuclease A, we show that it enables one to characterize the ample fluctuations in interdomain orientations expected to play an important functional role

Trends in template/fragment-free protein structure prediction

Predicting the structure of a protein from its amino acid sequence is a long-standing unsolved problem in computational biology. Its solution would be of both fundamental and practical importance as the gap between the number of known sequences and the number of experimentally solved structures widens rapidly. Currently, the most successful approaches are based on fragment/template reassembly. Lacking progress in template-free structure prediction calls for novel ideas and approaches. This article reviews trends in the development of physical and specific knowledge-based energy functions as well as sampling techniques for fragment-free structure prediction. Recent physical- and knowledge-based studies demonstrated that it is possible to sample and predict highly accurate protein structures without borrowing native fragments from known protein structures. These emerging approaches with fully flexible sampling have the potential to move the field forward

Determination of structural fluctuations of proteins from structure-based calculations of residual dipolar couplings

Residual dipolar couplings (RDCs) have the potential of providing detailed information about the conformational fluctuations of proteins. It is very challenging, however, to extract such information because of the complex relationship between RDCs and protein structures. A promising approach to decode this relationship involves structure-based calculations of the alignment tensors of protein conformations. By implementing this strategy to generate structural restraints in molecular dynamics simulations we show that it is possible to extract effectively the information provided by RDCs about the conformational fluctuations in the native states of proteins. The approach that we present can be used in a wide range of alignment media, including Pf1, charged bicelles and gels. The accuracy of the method is demonstrated by the analysis of the Q factors for RDCs not used as restraints in the calculations, which are significantly lower than those corresponding to existing high-resolution structures and structural ensembles, hence showing that we capture effectively the contributions to RDCs from conformational fluctuations. © 2012 Springer Science+Business Media B.V

Determination of conformational equilibria in proteins using residual dipolar couplings

In order to carry out their functions, proteins often undergo significant conformational fluctuations that enable them to interact with their partners. The accurate characterization of these motions is key in order to understand the mechanisms by which macromolecular recognition events take place. Nuclear magnetic resonance spectroscopy offers a variety of powerful methods to achieve this result. We discuss a method of using residual dipolar couplings as replica-averaged restraints in molecular dynamics simulations to determine large amplitude motions of proteins, including those involved in the conformational equilibria that are established through interconversions between different states. By applying this method to ribonuclease A, we show that it enables one to characterize the ample fluctuations in interdomain orientations expected to play an important functional role. © 2011 American Chemical Society

Characterization of the interdomain motions in hen lysozyme using residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations

Hen lysozyme is an enzyme characterized by the presence of two domains whose relative motions are involved in the mechanism of binding and release of the substrates. By using residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations, we characterize the breathing motions describing the interdomain fluctuations of this protein. We found that the ensemble of conformations that we determined spans the entire range of structures of hen lysozyme deposited in the Protein Data Bank, including both the free and bound states, suggesting that the thermal motions in the free state provide access to the structures populated upon binding. The approach that we present illustrates how the use of residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations makes it possible to explore conformational fluctuations of a relatively large amplitude in proteins. © 2013 American Chemical Society

Structures of the excited states of phospholamban and shifts in their populations upon phosphorylation

Phospholamban is an integral membrane protein that controls the calcium balance in cardiac muscle cells. As the function and regulation of this protein require the active involvement of low populated states in equilibrium with the native state, it is of great interest to acquire structural information about them. In this work, we calculate the conformations and populations of the ground state and the three main excited states of phospholamban by incorporating nuclear magnetic resonance residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations. We then provide a description of the manner in which phosphorylation at Ser16 modulates the activity of the protein by increasing the sizes of the populations of its excited states. These results demonstrate that the approach that we describe provides a detailed characterization of the different states of phospholamban that determine the function and regulation of this membrane protein. We anticipate that the knowledge of conformational ensembles enable the design of new dominant negative mutants of phospholamban by modulating the relative populations of its conformational substates. © 2013 American Chemical Society