The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains-the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)-flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the "coupled-allostery" model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.Severe acute respiratory syndrome (SARS) is the first pandemic of the 21st century that spread to multiple nations, with a fatality rate of ca. 8%. The disease is caused by a novel SARS-associated coronavirus (SARS-CoV) closely related to the group II coronaviruses, which include the human coronavirus OC43 and murine hepatitis virus (6, 18). Traditional antiviral treatments have had little success against SARS during the outbreak, and vaccines have yet to be developed (35).Coronaviruses are positive-sense single-stranded RNA (ssRNA) viruses. The coronavirus genomic RNA is encapsidated into a helical capsid by the nucleocapsid (N) protein, which is one of the most abundant coronavirus proteins (19). The N protein has nonspecific binding activity toward nucleic acids, including ssRNA, single-stranded DNA, and double-stranded DNA (33). It can also act as an RNA chaperone (39). However, the mechanism of binding of the N protein to nucleic acids is poorly understood.The SARS-CoV N protein is a homodimer composed of 422 amino acids (aa) in each chain. The N protein can be divided into two structural domains interspersed with disordered (unstructured) regions (Fig. 1A) (2). The N-terminal domain (NTD; also called RBD) serves as a putative RNA-binding domain, while the C-terminal domain (CTD; also called DD) is a dimerization domain (13,36). Both the NTD and the CTD bind to nucleic acids through electropositive regions on their surfaces (3, 13, 32). All coronaviruses share similar domain architectures at both the sequence and structure levels. No structure of N protein or any of its domains in complex with nucleic acids is ava...

The synchronized motions of inner core residues allosterically modulate the activity of the protein kinase A catalytic subunit.

Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a non-catalytic protein scaffold, we recently generated a novel RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely novel structure with dramatically enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.

We present an enhanced version of the FLAMEnGO (Fuzzy Logic Assignment of Methyl Group) software, a structure-based method to assign methyl group resonances in large proteins. FLAMEnGO utilizes a fuzzy logic algorithm coupled with Monte Carlo sampling to obtain a probability-based assignment of the methyl group resonances. As an input, FLAMEnGO requires the protein X-ray structure or an NMR structural ensemble with data such as methyl-methyl NOESY, paramagnetic relaxation enhancement (PRE), methine-methyl TOCSY data. Version 2.0 of this software (FLAMEnGO 2.0) has a user-friendly graphic interface and presents improved modules that enable the input of partial assignments and additional NMR restraints. We tested the performance of FLAMEnGO 2.0 on maltose binding protein (MBP) and the C-subunit of the cAMP-dependent protein kinase A (PKA-C). FLAMEnGO 2.0 can be used as a standalone method or to assist in the completion of partial resonance assignments and can be downloaded at www.chem.umn.edu/groups/veglia/forms/flamengo2-form.html.

KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-μs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.

Engineering and bioconjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multidomain protein may be complicated by undesirable side reactions to noninvolved domains. Furthermore, the advantages of segmental (or domain-specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods—expressed protein ligation, protein trans-splicing, and native chemical ligation—each have specific limitations. Here, we evaluated the use of different variants of Staphylococcus aureus sortase A for a range of ligation reactions and demonstrate that conditions can readily be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.

A new computational strategy is reported that provides a fast approximation of numerical solutions of differential equations in general. The method is demonstrated with the analysis of NMR adiabatic relaxation dispersion experiments to reveal biomolecular dynamics. When an analytical solution to the theoretical equations describing a physical process is not available, the new approach can significantly accelerate the computational speed of the conventional numerical integration up to 10(5) times. NMR adiabatic relaxation dispersion experiments enhanced with optimized proton-decoupled pulse sequences, although extremely powerful, have previously been refractory to quantitative analysis. Both simulations and experimental validation demonstrate detectable "slow" (microsecond to millisecond) conformational exchange rates from 10(2) to 10(5) s(-1). This greatly expanded time-scale range enables the characterization of a wide array of conformational fluctuations for individual residues, which correlate with biomolecular function and were previously inaccessible. Moreover, the new computational method can be potentially generalized for analysis of new types of relaxation dispersion experiments to characterize the various dynamics of biomolecular systems.

Structural biology often focuses primarily on three-dimensional structures of biological macromolecules, deposited in the Protein Data Bank (PDB). This resource is a remarkable entity for the worldwide scientific and medical communities, as well as the general public, as it is a growing translation into three-dimensional space of the vast information in genomic databases, e.g. GENBANK. There is, however, significantly more to understanding biological function than the three-dimensional co-ordinate space for ground-state structures of biomolecules. The vast array of biomolecules experiences natural dynamics, interconversion between multiple conformational states, and molecular recognition and allosteric events that play out on timescales ranging from picoseconds to seconds. This wide range of timescales demands ingenious and sophisticated experimental tools to sample and interpret these motions, thus enabling clearer insights into functional annotation of the PDB. NMR spectroscopy is unique in its ability to sample this range of timescales at atomic resolution and in physiologically relevant conditions using spin relaxation methods. The field is constantly expanding to provide new creative experiments, to yield more detailed coverage of timescales, and to broaden the power of interpretation and analysis methods. This review highlights the current state of the methodology and examines the extension of analysis tools for more complex experiments and dynamic models. The future for understanding protein dynamics is bright, and these extended tools bring greater compatibility with developments in computational molecular dynamics, all of which will further our understanding of biological molecular functions. These facets place NMR as a key component in integrated structural biology.