A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering protein.

TipA, a MerR family transcription factor from Streptomyces lividans, promotes antibiotic resistance by sequestering broad-spectrum thiopeptide-based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N-terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic-sequestration mechanisms. Here, combining equilibrium and time-resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and binding-competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding-competent state characterized by a partially structured N-terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic-sequestering proteins belonging to the MerR family.

Transient excited states of the metamorphic protein Mad2 and their implications for function.

The spindle checkpoint complex is a key surveillance mechanism in cell division that prevents premature separation of sister chromatids. Mad2 is an integral component of this spindle checkpoint complex that recognizes cognate substrates such as Mad1 and Cdc20 in its closed (C-Mad2) conformation by fastening a "seatbelt" around short peptide regions that bind to the substrate recognition site. Mad2 is also a metamorphic protein that adopts not only the fold found in C-Mad2, but also a structurally distinct open conformation (O-Mad2) which is incapable of binding substrates. Here, we show using chemical exchange saturation transfer (CEST) and relaxation dispersion (CPMG) NMR experiments that Mad2 transiently populates three other higher free energy states with millisecond lifetimes, two in equilibrium with C-Mad2 (E1 and E2) and one with O-Mad2 (E3). E1 is a mimic of substrate-bound C-Mad2 in which the N-terminus of one C-Mad2 molecule inserts into the seatbelt region of a second molecule of C-Mad2, providing a potential pathway for autoinhibition of C-Mad2. E2 is the "unbuckled" conformation of C-Mad2 that facilitates the triage of molecules along competing fold-switching and substrate binding pathways. The E3 conformation that coexists with O-Mad2 shows fluctuations at a hydrophobic lock that is required for stabilizing the O-Mad2 fold and we hypothesize that E3 represents an early intermediate on-pathway towards conversion to C-Mad2. Collectively, the NMR data highlight the rugged free energy landscape of Mad2 with multiple low-lying intermediates that interlink substrate-binding and fold-switching, and also emphasize the role of molecular dynamics in its function.

Measuring Hydroxyl Exchange Rates in Glycans Using a Synergistic Combination of Saturation Transfer and Relaxation Dispersion NMR.

Molecular recognition events mediated by glycans play pivotal roles in controlling the fate of diverse biological processes such as cellular communication and the immune response. The affinity of glycans for their target receptors is governed primarily by the hydrogen bonds formed by hydroxyl groups decorating the glycan surface. Hydroxyl exchange rate constants are therefore vital parameters that report on glycan structure and dynamics. Here we present a strategy for characterizing hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs a synergistic combination of 13C chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG) NMR methods. We show that the combination of CEST and CPMG experiments facilitates the sensitive detection of the small (∼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between protonated and deuterated states. This shift is leveraged for measuring site-specific kinetic H/D exchange rate constants as well as thermodynamic free energies of isotope fractionation. The CEST and CPMG modules are integrated with a selective J-cross-polarization scheme that provides the flexibility for rapid characterization of H/D exchange at a specific hydroxyl site. Moreover, our approach enables the precise isothermal measurement of hydroxyl exchange rate constants without the need for cumbersome isotope labeling. The H/D exchange rate constants of three different glycans assessed using this method highlight its potential for detecting transient intra- and intermolecular hydrogen bonds. In addition, the trends in H/D exchange rate constants establish site-specific steric accessibility as a key determinant of solvent exchange dynamics in glycans.

Probing excited state 1Hα chemical shifts in intrinsically disordered proteins with a triple resonance-based CEST experiment: Application to a disorder-to-order switch.

Over 40% of eukaryotic proteomes and 15% of bacterial proteomes are predicted to be intrinsically disordered based on their amino acid sequence. Intrinsically disordered proteins (IDPs) exist as heterogeneous ensembles of interconverting conformations and pose a challenge to the structure-function paradigm by apparently functioning without possessing stable structural elements. IDPs play a prominent role in biological processes involving extensive intermolecular interaction networks and their inherently dynamic nature facilitates their promiscuous interaction with multiple structurally diverse partner molecules. NMR spectroscopy has made pivotal contributions to our understanding of IDPs because of its unique ability to characterize heterogeneity at atomic resolution. NMR methods such as Chemical Exchange Saturation Transfer (CEST) and relaxation dispersion have enabled the detection of 'invisible' excited states in biomolecules which are transiently and sparsely populated, yet central for function. Here, we develop a 1Hα CEST pulse sequence which overcomes the resonance overlap problem in the 1Hα-13Cα plane of IDPs by taking advantage of the superior resolution in the 1H-15N correlation spectrum. In this sequence, magnetization is transferred after 1H CEST using a triple resonance coherence transfer pathway from 1Hα (i) to 1HN(i + 1) during which the 15N(t1) and 1HN(t2) are frequency labelled. This approach is integrated with spin state-selective CEST for eliminating spurious dips in CEST profiles resulting from dipolar cross-relaxation. We apply this sequence to determine the excited state 1Hα chemical shifts of the intrinsically disordered DNA binding domain (CytRN) of the bacterial cytidine repressor (CytR), which transiently acquires a functional globally folded conformation. The structure of the excited state, calculated using 1Hα chemical shifts in conjunction with other excited state NMR restraints, is a three-helix bundle incorporating a helix-turn-helix motif that is vital for binding DNA.

Studying micro to millisecond protein dynamics using simple amide 15N CEST experiments supplemented with major-state R2 and visible peak-position constraints.

Over the last decade amide 15N CEST experiments have emerged as a popular tool to study protein dynamics that involves exchange between a 'visible' major state and sparsely populated 'invisible' minor states. Although initially introduced to study exchange between states that are in slow exchange with each other (typical exchange rates of, 10 to 400 s-1), they are now used to study interconversion between states on the intermediate to fast exchange timescale while still using low to moderate (5 to 350 Hz) 'saturating' B1 fields. The 15N CEST experiment is very sensitive to exchange as the exchange delay TEX can be quite long (~0.5 s) allowing for a large number of exchange events to occur making it a very powerful tool to detect minor sates populated ([Formula: see text]) to as low as 1%. When systems are in fast exchange and the 15N CEST data has to be described using a model that contains exchange, the exchange parameters are often poorly defined because the [Formula: see text] versus [Formula: see text] and [Formula: see text] versus exchange rate ([Formula: see text]) plots can be quite flat with shallow or no minima and the analysis of such 15N CEST data can lead to wrong estimates of the exchange parameters due to the presence of 'spurious' minima. Here we show that the inclusion of experimentally derived constraints on the intrinsic transverse relaxation rates and the inclusion of visible state peak-positions during the analysis of amide 15N CEST data acquired with moderate B1 values (~50 to ~350 Hz) results in convincing minima in the [Formula: see text] versus [Formula: see text] and the [Formula: see text] versus [Formula: see text] plots even when exchange occurs on the 100 µs timescale. The utility of this strategy is demonstrated on the fast-folding Bacillus stearothermophilus peripheral subunit binding domain that folds with a rate constant ~104 s-1. Here the analysis of 15N CEST data alone results in [Formula: see text] versus [Formula: see text] and [Formula: see text] versus [Formula: see text] plots that contain shallow minima, but the inclusion of visible-state peak positions and restraints on the intrinsic transverse relaxation rates of both states during the analysis of the 15N CEST data results in pronounced minima in the [Formula: see text] versus [Formula: see text] and [Formula: see text] versus [Formula: see text] plots and precise exchange parameters even in the fast exchange regime ([Formula: see text]~5). Using this strategy we find that the folding rate constant of PSBD is invariant (~10,500 s-1) from 33.2 to 42.9 °C while the unfolding rates (~70 to ~500 s-1) and unfolded state populations (~0.7 to ~4.3%) increase with temperature. The results presented here show that protein dynamics occurring on the 10 to 104 s-1 timescale can be studied using amide 15N CEST experiments.

Structure and Carbohydrate Recognition by the Nonmitogenic Lectin Horcolin.

Lectins are sugar-binding proteins that have shown considerable promise as antiviral agents because of their ability to interact with envelope glycoproteins present on the surface of viruses such as HIV-1. However, their therapeutic potential has been compromised by their mitogenicity that stimulates uncontrolled division of T-lymphocytes. Horcolin, a member of the jacalin family of lectins, tightly binds the HIV-1 envelope glycoprotein gp120 and neutralizes HIV-1 particles but is nonmitogenic. In this report, we combine X-ray crystallography and NMR spectroscopy to obtain atomic-resolution insights into the structure of horcolin and the molecular basis for its carbohydrate recognition. Each protomer of the horcolin dimer adopts a canonical ß-prism I fold with three Greek key motifs and carries two carbohydrate-binding sites. The carbohydrate molecule binds in a negatively charged pocket and is stabilized by backbone and side chain hydrogen bonds to conserved residues in the ligand-binding loop. NMR titrations reveal a two-site binding mode and equilibrium dissociation constants for the two binding sites determined from two-dimensional (2D) lineshape modeling are 4-fold different. Single-binding-site variants of horcolin confirm the dichotomy in binding sites and suggest that there is allosteric communication between the two sites. An analysis of the horcolin structure shows a network of hydrogen bonds linking the two carbohydrate-binding sites directly and through a secondary binding site, and this coupling between the two sites is expected to assume importance in the interaction of horcolin with high-mannose glycans found on viral envelope glycoproteins.

Effects of maturation on the conformational free-energy landscape of SOD1.

Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer's, Parkinson's, and Huntington's diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components. We have shown previously that immature SOD1, lacking bound Cu and Zn metal ions and the intrasubunit disulfide bond (apoSOD12SH), has a rugged free-energy surface (FES) and exchanges with four other conformations (excited states) that have millisecond lifetimes and sparse populations on the order of a few percent. Here, we examine further states of SOD1 along its maturation pathway, as well as those off-pathway resulting from metal loss that have been observed in proteinaceous inclusions. Metallation and disulfide bond formation lead to structural transformations including local ordering of the electrostatic loop and native dimerization that are observed in rare conformers of apoSOD12SH; thus, SOD1 maturation may occur via a population-switch mechanism whereby posttranslational modifications select for preexisting structures on the FES. Metallation and oxidation of SOD1 stabilize the native, mature conformation and decrease the number of detected excited conformational states, suggesting that it is the immature forms of the protein that contribute to misfolded conformations in vivo rather than the highly stable enzymatically active dimer.

Characterizing Post-Translational Modifications and Their Effects on Protein Conformation Using NMR Spectroscopy.

The diversity of the cellular proteome substantially exceeds the number of genes coded by the DNA of an organism because one or more residues in a majority of eukaryotic proteins are post-translationally modified (PTM) by the covalent conjugation of specific chemical groups. We now know that PTMs alter protein conformation and function in ways that are not entirely understood at the molecular level. NMR spectroscopy has been particularly successful as an analytical tool in elucidating the themes underlying the structural role of PTMs. In this Perspective, we focus on the NMR-based characterization of three abundant PTMs: phosphorylation, acetylation, and glycosylation. We detail NMR methods that have found success in detecting these modifications at a site-specific level. We also highlight NMR studies that have mapped the conformational changes ensuing from these PTMs as well as evaluated their relation to function. The NMR toolbox is expanding rapidly with experiments available to probe not only the average structure of biomolecules but also how this structure changes with time on time scales ranging from picoseconds to seconds. The atomic resolution insights into the biomolecular structure, dynamics, and mechanism accessible from NMR spectroscopy ensure that NMR will continue to be at the forefront of research in the structural biology of PTMs.

Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation.

Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid "membraneless organelles" that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, [Formula: see text], diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing [Formula: see text] to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of [Formula: see text] have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in [Formula: see text] are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in [Formula: see text] are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.

Engineering Order and Cooperativity in a Disordered Protein.

Structural disorder in proteins arises from a complex interplay between weak hydrophobicity and unfavorable electrostatic interactions. The extent to which the hydrophobic effect contributes to the unique and compact native state of proteins is, however, confounded by large compensation between multiple entropic and energetic terms. Here we show that protein structural order and cooperativity arise as emergent properties upon hydrophobic substitutions in a disordered system with non-intuitive effects on folding and function. Aided by sequence-structure analysis, equilibrium, and kinetic spectroscopic studies, we engineer two hydrophobic mutations in the disordered DNA-binding domain of CytR that act synergistically, but not in isolation, to promote structure, compactness, and stability. The double mutant, with properties of a fully ordered domain, exhibits weak cooperativity with a complex and rugged conformational landscape. The mutant, however, binds cognate DNA with an affinity only marginally higher than that of the wild type, though nontrivial differences are observed in the binding to noncognate DNA. Our work provides direct experimental evidence of the dominant role of non-additive hydrophobic effects in shaping the molecular evolution of order in disordered proteins and vice versa, which could be generalized to even folded proteins with implications for protein design and functional manipulation.

Hsp70 biases the folding pathways of client proteins.

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a (1)H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.

Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that, in some cases, has been linked with mutations to the antioxidant metalloenzyme superoxide dismutase (SOD1). Although the mature form of this enzyme is highly stable and resistant to aggregation, the most immature form, lacking metal and a stabilizing intrasubunit disulfide bond, apoSOD12SH, is dynamic and hypothesized to be a major cause of toxicity in vivo. Previous solution NMR studies of wild-type apoSOD12SH have shown that the ground state interconverts with a series of sparsely populated and transiently formed conformers, some of which have aberrant nonnative structures. Here, we study seven disease mutants of apoSOD12SH and characterize their free energy landscapes as a first step in understanding the initial stages of disease progression and, more generally, to evaluate the plasticity of low-lying protein conformational states. The mutations lead to little change in the structures and dynamics of the ground states of the mutant proteins. By contrast, the numbers of low-lying excited states that are accessible to each of the disease mutants can vary significantly, with additional conformers accessed in some cases. Our study suggests that the diversity of these structures can provide alternate interaction motifs for different mutants, establishing additional pathways for new and often aberrant intra- and intermolecular contacts. Further, it emphasizes the potential importance of conformationally excited states in directing both folding and misfolding processes.

Mapping the conformation of a client protein through the Hsp70 functional cycle.

The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.

Heterogeneous binding of the SH3 client protein to the DnaK molecular chaperone.

The molecular chaperone heat shock protein 70 (Hsp70) plays a vital role in cellular processes, including protein folding and assembly, and helps prevent aggregation under physiological and stress-related conditions. Although the structural changes undergone by full-length client proteins upon interaction with DnaK (i.e., Escherichia coli Hsp70) are fundamental to understand chaperone-mediated protein folding, these changes are still largely unexplored. Here, we show that multiple conformations of the SRC homology 3 domain (SH3) client protein interact with the ADP-bound form of the DnaK chaperone. Chaperone-bound SH3 is largely unstructured yet distinct from the unfolded state in the absence of DnaK. The bound client protein shares a highly flexible N terminus and multiple slowly interconverting conformations in different parts of the sequence. In all, there is significant structural and dynamical heterogeneity in the DnaK-bound client protein, revealing that proteins may undergo some conformational sampling while chaperone-bound. This result is important because it shows that the surface of the Hsp70 chaperone provides an aggregation-free environment able to support part of the search for the native state.

Measuring Diffusion Constants of Invisible Protein Conformers by Triple-Quantum 1 H CPMG Relaxation Dispersion.

Proteins are not locked in a single structure but often interconvert with other conformers that are critical for function. When such conformers are sparsely populated and transiently formed they become invisible to routine biophysical methods, however they can be studied in detail by NMR spin-relaxation experiments. Few experiments are available in the NMR toolkit, however, for characterizing the hydrodynamic properties of invisible states. Herein we describe a CPMG-based experiment for measuring translational diffusion constants of invisible states using a pulsed-field gradient approach that exploits methyl 1 H triple-quantum coherences. An example, involving diffusion of a sparsely populated and hence invisible unfolded protein ensemble is presented, without the need for the addition of denaturants that tend to destroy weak interactions that can be involved in stabilizing residual structure in the unfolded state.

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. We show that in solution and at millimolar concentrations profilin-1 is predominantly monomeric. However, fits of concentration-dependent relaxation data are consistent with the formation of a higher-order oligomer that is generated via a multistep process. Together with crystallographic data for profilin-2, a homologue of the protein studied here, our results suggest that profilin-1 forms a sparsely populated tetrameric conformer in solution.

Probing conformational dynamics in biomolecules via chemical exchange saturation transfer: a primer.

Although Chemical Exchange Saturation Transfer (CEST) type NMR experiments have been used to study chemical exchange processes in molecules since the early 1960s, there has been renewed interest in the past several years in using this approach to study biomolecular conformational dynamics. The methodology is particularly powerful for the study of sparsely populated, transiently formed conformers that are recalcitrant to investigation using traditional biophysical tools, and it is complementary to relaxation dispersion and magnetization transfer experiments that have traditionally been used to study chemical exchange processes. Here we discuss the concepts behind the CEST experiment, focusing on practical aspects as well, we review some of the pulse sequences that have been developed to characterize protein and RNA conformational dynamics, and we discuss a number of examples where the CEST methodology has provided important insights into the role of dynamics in biomolecular function.

An enhanced sensitivity methyl 1H triple-quantum pulse scheme for measuring diffusion constants of macromolecules.

We present a pulse scheme that exploits methyl 1H triple-quantum (TQ) coherences for the measurement of diffusion rates of slowly diffusing molecules in solution. It is based on the well-known stimulated echo experiment, with encoding and decoding of TQ coherences. The size of quantifiable diffusion coefficients is thus lowered by an order of magnitude with respect to single-quantum (SQ) approaches. Notably, the sensitivity of the scheme is high, approximately ¾ that of the corresponding single quantum experiment, neglecting relaxation losses, and on the order of a factor of 4 more sensitive than a previously published sequence for AX3 spin systems (Zheng et al. in JMR 198:271-274, 2009) for molecules that are only 13C labeled at the methyl carbon position. Diffusion coefficients measured from TQ- and SQ-based experiments recorded on a range of protein samples are in excellent agreement. We present an application of this technique to the study of phase-separated proteins where protein concentrations in the condensed phase can exceed 400 mg/mL, diffusion coefficients can be as low as ~10-9 cm2s-1 and traditional SQ experiments fail.

Understanding the mechanism of proteasome 20S core particle gating.

The 20S core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. Regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. Although atomic resolution models of both open and closed states of the proteasome have been elucidated, the mechanism by which gates exchange between these states remains to be understood. Here, this is investigated by using magnetization transfer NMR spectroscopy focusing on the 20S proteasome core particle from Thermoplasma acidophilum. We show from viscosity-dependent proteasome gating kinetics that frictional forces originating from random solvent motions are critical for driving the gating process. Notably, a small effective hydrodynamic radius (EHR; <4Å) is obtained, providing a picture in which gate exchange proceeds through many steps involving only very small segment sizes. A small EHR further suggests that the kinetics of gate interconversion will not be affected appreciably by large viscogens, such as macromolecules found in the cell, so long as they are inert. Indeed, measurements in cell lysate reveal that the gate interconversion rate decreases only slightly, demonstrating that controlled studies in vitro provide an excellent starting point for understanding regulation of 20S core particle function in complex, biologically relevant environments.

Separating Dipolar and Chemical Exchange Magnetization Transfer Processes in 1 H-CEST.

An amide 1 H-Chemical Exchange Saturation Transfer (CEST) experiment is presented for studies of conformational exchange in proteins. The approach, exploiting spin-state-selective magnetization transfer, completely suppresses undesired NOE-based dips in CEST profiles so that chemical exchange processes can be studied. The methodology is demonstrated with applications involving proteins that interconvert on the millisecond timescale between major and invisible minor states, and accurate amide 1 H chemical shifts of the minor conformer are obtained in each case. The spin-state-selective magnetization transfer approach offers unique possibilities for quantitative studies of protein exchange through 1 H-CEST.