David Ban

Kinetics of Conformational Sampling in Ubiquitin

Molecular recognition plays a central role in many biological processes. For enzymatic reactions and slow protein–protein recognition events, turn-over rates and on-rates in the millisecond-to-second time scale have been connected to internal protein dynamics detected with atomic resolution by NMR spectroscopy, and in particular conformational sampling could be established as a mechanism for enzyme–substrate and protein–protein recognition. Recent theoretical studies indicate that faster rates of conformational interconversion in the microsecond time scale might limit on-rates for protein–protein recognition. However experimental proofs were lacking so far, mainly because such rates could not be determined accurately enough and kinetic experiments in the microsecond time range are difficult to perform. Nevertheless, for proteins and TAR-RNA, recent studies based on residual dipolar couplings (RDCs) and other NMR spectroscopy techniques have detected substantial internal dynamics in a time window from the rotational correlation time tc (one-digit nanoseconds) to approximately 50 ms, called the supra-tc window in the following. However, the exact rates of internal dynamics within this four orders of magnitude wide time window could not be determined. Supra-tc dynamics in ubiquitin [9] and TAR-RNA could be connected to the conformational sampling required for molecular recognition. While the amplitudes of motions have been indirectly detected by RDCs and characterized in great detail, it has so far been impossible to directly observe these motions and to determine the exact rate of these supra-tc motions. In contrast, conformational sampling in enzymes occurs on a time scale that is 100 to 1000 times slower than supra-tc dynamics and therefore NMR relaxation dispersion (RD) techniques have been able to establish the functional link to enzyme kinetics with atomic resolution at physiological conditions. 5] However, for technical reasons, RD is not sensitive to motion faster than approximately 50 ms (RD window) and therefore does not access motion in the supra-tc window at room temperature. Here we determine the rate of interconversion between conformers of free ubiquitin by a combination of NMR RD experiments in super-cooled solution and dielectric relaxation spectroscopy (DR). Furthermore, we corroborate the motional amplitudes in the RDC-derived ensembles quantitatively with the observed amplitudes of RD and DR. The methods utilized herein can be used to directly study protein dynamics in a time range that was previously inaccessible. Significant motional amplitude in the supra-tc window has been observed using RDC measurements, and was connected to the conformational sampling for a protein in the ground

Exceeding the limit of dynamics studies on biomolecules using high spin-lock field strengths with a cryogenically cooled probehead.

Internal motions in the microsecond timescale have been proposed to play an active part in a proteins biological function. Nuclear magnetic resonance (NMR) relaxation dispersion is a robust method sensitive to this timescale with atomic resolution. However, due to technical limitations, the observation of motions faster than ∼40 μs for ¹⁵N nuclei was not possible. We show that with a cryogenically cooled NMR probehead, a high spin-lock field strength can be generated that is able to detect motions as fast as 25 μs. We apply this high spin-lock field strength in an NMR experiment used for characterizing dynamical processes. An on-resonance rotating-frame transverse relaxation experiment was implemented that allows for the detection of a 25 μs process from a dispersion curve, and transverse relaxation rates were compared at low and high spin-lock field strengths showing that at high field strengths contributions from chemical exchange with lifetimes up to 25 μs can be removed. Due to the increase in sensitivity towards fast motion, relaxation dispersion for a residue that undergoes smaller chemical shift variations due to dynamics was identified. This technique reduces the previously inaccessible window between the correlation time and the relaxation dispersion window that covers four orders of magnitude by a factor of 2.

A designed conformational shift to control protein binding specificity.

In a conformational selection scenario, manipulating the populations of binding-competent states should be expected to affect protein binding. We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground-state ensemble between open and closed substates that have a similar population in the wild-type protein. Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin’s binding specificity and hence its function. Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding-competent substates.

Measuring Dynamic and Kinetic Information in the Previously Inaccessible Supra-tc Window of Nanoseconds to Microseconds by Solution NMR Spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool that has enabled experimentalists to characterize molecular dynamics and kinetics spanning a wide range of time-scales from picoseconds to days. This review focuses on addressing the previously inaccessible supra-τc window (defined as τc < supra-τc < 40 μs; in which τc is the overall tumbling time of a molecule) from the perspective of local inter-nuclear vector dynamics extracted from residual dipolar couplings (RDCs) and from the perspective of conformational exchange captured by relaxation dispersion measurements (RD). The goal of the first section is to present a detailed analysis of how to extract protein dynamics encoded in RDCs and how to relate this information to protein functionality within the previously inaccessible supra-τc window. In the second section, the current state of the art for RD is analyzed, as well as the considerable progress toward pushing the sensitivity of RD further into the supra-τc scale by up to a factor of two (motion up to 25 μs). From the data obtained with these techniques and methodology, the importance of the supra-τc scale for protein function and molecular recognition is becoming increasingly clearer as the connection between motion on the supra-τc scale and protein functionality from the experimental side is further strengthened with results from molecular dynamics simulations.

Allosteric switch regulates protein–protein binding through collective motion.

Significance Within biological molecules, a change in shape at one site affecting a distant site is called allostery, and is a process critical for sustaining life. At the atomic level, the means of communication is often not well understood. We identify a previously unidentified way for allostery to occur through collective motion of an entire protein structure. Together with newly developed computational algorithms, we determine atomic structures representing this previously invisible allosteric motion. This allosteric network is shown to be an intrinsic property of the protein and important for enabling binding to different protein partners. The nature of this motion, along with the tools we developed to detect it, should prove invaluable for understanding living organisms and developing new therapeutics. Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.

ORIUM: optimized RDC-based Iterative and Unified Model-free analysis.

Residual dipolar couplings (RDCs) are NMR parameters that provide both structural and dynamic information concerning inter-nuclear vectors, such as N–HN and Cα–Hα bonds within the protein backbone. Two approaches for extracting this information from RDCs are the model free analysis (MFA) (Meiler et al. in J Am Chem Soc 123:6098–6107, 2001; Peti et al. in J Am Chem Soc 124:5822–5833, 2002) and the direct interpretation of dipolar couplings (DIDCs) (Tolman in J Am Chem Soc 124:12020–12030, 2002). Both methods have been incorporated into iterative schemes, namely the self-consistent RDC based MFA (SCRM) (Lakomek et al. in J Biomol NMR 41:139–155, 2008) and iterative DIDC (Yao et al. in J Phys Chem B 112:6045–6056, 2008), with the goal of removing the influence of structural noise in the MFA and DIDC formulations. Here, we report a new iterative procedure entitled Optimized RDC-based Iterative and Unified Model-free analysis (ORIUM). ORIUM unifies theoretical concepts developed in the MFA, SCRM, and DIDC methods to construct a computationally less demanding approach to determine these structural and dynamic parameters. In all schemes, dynamic averaging reduces the actual magnitude of the alignment tensors complicating the determination of the absolute values for the generalized order parameters. To readdress this scaling issue that has been previously investigated (Lakomek et al. in J Biomol NMR 41:139–155, 2008; Salmon et al. in Angew Chem Int Edit 48:4154–4157, 2009), a new method is presented using only RDC data to establish a lower bound on protein motion, bypassing the requirement of Lipari–Szabo order parameters. ORIUM and the new scaling procedure are applied to the proteins ubiquitin and the third immunoglobulin domain of protein G (GB3). Our results indicate good agreement with the SCRM and iterative DIDC approaches and signify the general applicability of ORIUM and the proposed scaling for the extraction of inter-nuclear vector structural and dynamic content.

Kinetics of the Antibody Recognition Site in the Third IgG‐Binding Domain of Protein G

Protein dynamics occurring on a wide range of timescales play a crucial role in governing protein function. Particularly, motions between the globular rotational correlation time (τc ) and 40 μs (supra-τc window), strongly influence molecular recognition. This supra-τc window was previously hidden, owing to a lack of experimental methods. Recently, we have developed a high-power relaxation dispersion (RD) experiment for measuring kinetics as fast as 4 μs. For the first time, this method, performed under super-cooled conditions, enabled us to detect a global motion in the first β-turn of the third IgG-binding domain of protein G (GB3), which was extrapolated to 371±115 ns at 310 K. Furthermore, the same residues show the plasticity in the model-free residual dipolar coupling (RDC) order parameters and in an ensemble encoding the supra-τc dynamics. This β-turn is involved in antibody binding, exhibiting the potential link of the observed supra-τc motion with molecular recognition.

High-power 1H composite pulse decoupling provides artifact free exchange-mediated saturation transfer (EST) experiments

Exchange-mediated saturation transfer (EST) provides critical information regarding dynamics of molecules. In typical applications EST is studied by either scanning a wide range of (15)N chemical shift offsets where the applied (15)N irradiation field strength is on the order of hundreds of Hertz or, scanning a narrow range of (15)N chemical shift offsets where the applied (15)N irradiation field-strength is on the order of tens of Hertz during the EST period. The (1)H decoupling during the EST delay is critical as incomplete decoupling causes broadening of the EST profile, which could possibly result in inaccuracies of the extracted kinetic parameters and transverse relaxation rates. Currently two different (1)H decoupling schemes have been employed, intermittently applied 180° pulses and composite-pulse-decoupling (CPD), for situations where a wide range, or narrow range of (15)N chemical shift offsets are scanned, respectively. We show that high-power CPD provides artifact free EST experiments, which can be universally implemented regardless of the offset range or irradiation field-strengths.

1H, 13C and 15N resonance assignment of human guanylate kinase

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain 1H, 13C, and 15N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.

Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I

Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44-56) and more weakly binding PAR3G (44-56) could already interact with pro-ABE I on prothrombin. 1H-15N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual 15N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.