Dynamic conformational equilibria in the active states of KRAS and NRAS.

The design of potent RAS inhibitors benefits from a molecular understanding of the dynamics in KRAS and NRAS and their oncogenic mutants. Here we characterize switch-1 dynamics in GTP-state KRAS and NRAS by P NMR, by N relaxation dispersion NMR, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics simulations. In GMPPNP-bound KRAS and NRAS, we see the co-existence of two conformational states, corresponding to an "inactive" state-1 and an "active" state-2, as previously reported.

Design of amyloidogenic peptide traps.

Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins.

Exploiting conformational dynamics to modulate the function of designed proteins.

With the recent success in calculating protein structures from amino acid sequences using artificial intelligence-based algorithms, an important next step is to decipher how dynamics is encoded by the primary protein sequence so as to better predict function. Such dynamics information is critical for protein design, where strategies could then focus not only on sequences that fold into particular structures that perform a given task, but would also include low-lying excited protein states that could influence the function of the designed protein. Herein, we illustrate the importance of dynamics in modulating the function of C34, a designed α/β protein that captures β-strands of target ligands and is a member of a family of proteins designed to sequester β-strands and β hairpins of aggregation-prone molecules that lead to a variety of pathologies.

Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson's disease.

Accumulation of α-synuclein into toxic oligomers or fibrils is implicated in dopaminergic neurodegeneration in Parkinson's disease. Here we performed a high-throughput, proteome-wide peptide screen to identify protein-protein interaction inhibitors that reduce α-synuclein oligomer levels and their associated cytotoxicity. We find that the most potent peptide inhibitor disrupts the direct interaction between the C-terminal region of α-synuclein and CHarged Multivesicular body Protein 2B (CHMP2B), a component of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III).

Opening of a cryptic pocket in β-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop.

A methyl-TROSY approach for NMR studies of high-molecular-weight DNA with application to the nucleosome core particle.

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)-based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics.

Confronting the Invisible: Assignment of Protein H Chemical Shifts in Cases of Extreme Broadening.

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where H spectra cannot be obtained.

An allosteric switch regulates ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR.

The 300-kDa ClpP1P2 protease from collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function.

Exploring long-range cooperativity in the 20S proteasome core particle from using methyl-TROSY-based NMR.

The 20S core particle (CP) proteasome is a molecular assembly catalyzing the degradation of misfolded proteins or proteins no longer required for function. It is composed of four stacked heptameric rings that form a barrel-like structure, sequestering proteolytic sites inside its lumen. Proteasome function is regulated by gates derived from the termini of α-rings and through binding of regulatory particles (RPs) to one or both ends of the barrel.

The Role of Protein Thermodynamics and Primary Structure in Fibrillogenesis of Variable Domains from Immunoglobulin Light Chains.

Immunoglobulin light-chain amyloidosis is a protein aggregation disease that leads to proteinaceous deposits in a variety of organs in the body and, if untreated, ultimately results in death. The mechanisms by which light-chain aggregation occurs are not well understood. Here we have used solution NMR spectroscopy and biophysical studies to probe immunoglobulin variable domain λV6-57 V aggregation, a process that appears to drive the degenerative phenotypes in amyloidosis patients.

Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs.

Role of domain interactions in the aggregation of full-length immunoglobulin light chains.

Amyloid light-chain (LC) amyloidosis is a protein misfolding disease in which the aggregation of an overexpressed antibody LC from a clonal plasma cell leads to organ toxicity and patient death if left untreated. While the overall dimeric architecture of LC molecules is established, with each LC composed of variable (V) and constant (C) domains, the relative contributions of LC domain-domain interfaces and intrinsic domain stabilities to protection against LC aggregation are not well understood. To address these topics we have engineered a number of domain-destabilized LC mutants and used solution NMR spectroscopy to characterize their structural properties and intrinsic stabilities.

Exploiting conformational plasticity in the AAA+ protein VCP/p97 to modify function.

p97/VCP, a member of the AAA+ (ATPases associated with diverse cellular activities) family of proteins, is implicated in the etiology of a group of degenerative diseases affecting bone and muscle tissue as well as the central nervous system. Methyl-TROSY-based NMR studies have previously revealed how disease-causing mutations deregulate a subtle dynamic conformational equilibrium involving the N-terminal domain (NTD) with implications for the binding of certain adaptors, providing insight into how disease mutations lead to abnormal function. Herein the conformational plasticity of the p97 system is explored in an attempt to identify hotspots that can serve as targets for restoring function in disease mutants by shifting the position of the NTD back to its wild-type location.

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets.

Self-Assembly of Human Profilin-1 Detected by Carr-Purcell-Meiboom-Gill Nuclear Magnetic Resonance (CPMG NMR) Spectroscopy.

Protein oligomerization in the cell has important implications for both health and disease, and an understanding of the mechanisms by which proteins can self-associate is, therefore, of critical interest. Initial stages of the oligomerization process can be hard to detect, as they often involve the formation of sparsely populated and transient states that are difficult to characterize by standard biophysical approaches. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we study the oligomerization of human profilin-1, a protein that regulates the polymerization of actin.

Quantitative measurement of exchange dynamics in proteins via (13)C relaxation dispersion of (13)CHD2-labeled samples.

Methyl groups have emerged as powerful probes of protein dynamics with timescales from picoseconds to seconds. Typically, studies involving high molecular weight complexes exploit (13)CH3- or (13)CHD2-labeling in otherwise highly deuterated proteins. The (13)CHD2 label offers the unique advantage of providing (13)C, (1)H and (2)H spin probes, however a disadvantage has been the lack of an experiment to record (13)C Carr-Purcell-Meiboom-Gill relaxation dispersion that monitors millisecond time-scale dynamics, implicated in a wide range of biological processes.

ClpB N-terminal domain plays a regulatory role in protein disaggregation.

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive.

(13)CHD2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins.

An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of (13)CHD2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble.

Measuring hydrogen exchange in proteins by selective water saturation in (1)H- (15)N SOFAST/BEST-type experiments: advantages and limitations.

HET(ex)-SOFAST NMR (Schanda et al. in J Biomol NMR 33:199-211, 2006) has been proposed some years ago as a fast and sensitive method for semi-quantitative measurement of site-specific amide-water hydrogen exchange effects along the backbone of proteins. Here we extend this concept to BEST readout sequences that provide a better resolution at the expense of some loss in sensitivity.

Fast real-time NMR methods for characterizing short-lived molecular states.

The characterization of both the structure and the conformational dynamics of biological macromolecules, namely proteins and nucleic acids, is required for understanding the molecular mechanisms underlying physiological function and disease. Molecular dynamics involves the transient departure from the ground-state structures to populate short-lived excited state conformations that can play important functional roles. Real-time multi-dimensional NMR spectroscopy represents a unique tool for investigating dynamic molecular processes occurring on time scales of seconds or longer, providing atomic-resolution information about short-lived states.

Real-time NMR characterization of structure and dynamics in a transiently populated protein folding intermediate.

Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding.

Single-shot NMR measurement of protein unfolding landscapes.

The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine-Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is (15)N-labelled.

Determining the energy landscape of proteins by a fast isotope exchange NMR approach.

We present a new and efficient NMR method, BLUU-Tramp (Biophysics Laboratory University of Udine temperature ramp), for the collection of hydrogen-deuterium exchange experiments as a function of time and temperature for small and medium-sized proteins. Exchange rates can be determined to extract the underlying thermodynamic equilibrium or kinetic parameters by sampling hundreds of points over a virtually continuous temperature ramp. Data are acquired in a single experimental session that lasts some 20-60 h, depending on the thermal stability of the protein.