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Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites., (© 2024. The Author(s).)

Strategies that mimic the spatial complexity of natural tissues can provide cellular scaffolds to probe fundamental questions in cell biology and offer new materials for regenerative medicine. Here, the authors demonstrate a light-guided patterning platform that uses natural engineered extracellular matrix (ECM) proteins as a substrate to program cellular behaviors. A photocaged diene which undergoes Diels-Alder-based click chemistry upon uncaging with 365 nm light is utilized. By interfacing with commercially available maleimide dienophiles, patterning of common ECM proteins (collagen, fibronectin Matrigel, laminin) with readily purchased functional small molecules and growth factors is achieved. Finally, the use of this platform to spatially control ERK activity and migration in mammalian cells is highlighted, demonstrating programmable cell behavior through patterned chemical modification of natural ECM., (© 2023 Wiley-VCH GmbH.)

Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence., (© 2022. The Author(s).)

Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use β-phosphoglucomutase (βPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. βPGM catalyzes the isomerization of β-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. βPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported β-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

The COVID-19 pandemic has taken a devastating human toll worldwide. The development of impactful guidelines and measures for controlling the COVID-19 pandemic requires continuous and widespread testing of suspected cases and their contacts through accurate, accessible, and reliable methods for SARS-CoV-2 detection. Here we describe a CRISPR-Cas13-based method for the detection of SARS-CoV-2. The assay is called CREST (Cas13-based, rugged, equitable, scalable testing), and is specific, sensitive, and highly accessible. As such, CREST may provide a low-cost and dependable alternative for SARS-CoV-2 surveillance. © 2022 Wiley Periodicals LLC. Basic Protocol: Cas13-ased detection of SARS-CoV-2 genetic material using a real-time PCR detection system Alternate Protocol: Cas13-based detection of SARS-CoV-2 genetic material using a fluorescence viewer Support Protocol 1: LwaCas13a purification Support Protocol 2: In vitro transcription of synthetic targets., (© 2022 Wiley Periodicals LLC.)

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance., (Copyright © 2021 Rauch et al.)

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21-24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.

Nuclear magnetic resonance (NMR) is a powerful tool to study three-dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N 1 and N 2 and plus another folded conformation, I, in which some amide group signals of residues 33-41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well-converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure-induced loss of backbone amide group signals at residues 28, 33, 36, and 39-41 like the wild-type, with a similar but smaller effect on CαH and CβH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N 2 conformation, although the amide groups at residues 33-41 have more heterogeneous conformations and more contact with water, which differ from the N 1 and N 2 states. The PRE-assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations., (© 2019 The Protein Society.)

β-Phosphoglucomutase (βPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of β-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a β-glucose 1,6-bisphosphate intermediate. Substrate-free βPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into βPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free βPGM WT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report 1 H, 15 N and 13 C backbone resonance assignments of the P146A variant (βPGM P146A ) in its substrate-free form, where the K145-A146 peptide bond adopts a trans conformation in contrast to all crystal structures of βPGM WT , where the K145-P146 peptide bond is cis. In βPGM P146A millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect βPGM crystal structures, and a chemical shift comparison between substrate-free βPGM P146A and βPGM WT confirms that the solution conformations are very similar, except for the D137-A147 loop. Hence, the isomerisation state of the 145-146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12-T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114-S116), and the D137-A147 loop (V141-A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic Mg II ion and the L44-L53 loop responsible for substrate discrimination.

Catechol- O -methyltransferase (COMT) is a model S-adenosyl-l-methionine (SAM) dependent methyl transferase, which catalyzes the methylation of catecholamine neurotransmitters such as dopamine in the primary pathway of neurotransmitter deactivation in animals. Despite extensive study, there is no consensus view of the physical basis of catalysis in COMT. Further progress requires experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of positions along the reaction coordinate. Here we establish that sinefungin, a fungal-derived inhibitor of SAM-dependent enzymes that possess transition state-like charge on the transferring group, can be used as a transition state analog of COMT when combined with a catechol. X-ray crystal structures and NMR backbone assignments of the ternary complexes of the soluble form of human COMT containing dinitrocatechol, Mg 2+ and SAM or sinefungin were determined. Comparison and further analysis with the aid of density functional theory calculations and molecular dynamics simulations provides evidence for active site "compaction", which is driven by electrostatic stabilization between the transferring methyl group and "equatorial" active site residues that are orthogonal to the donor-acceptor (pseudo reaction) coordinate. We propose that upon catecholamine binding and subsequent proton transfer to Lys 144, the enzyme becomes geometrically preorganized, with little further movement along the donor-acceptor coordinate required for methyl transfer. Catalysis is then largely facilitated through stabilization of the developing charge on the transferring methyl group via "equatorial" H-bonding and electrostatic interactions orthogonal to the donor-acceptor coordinate., Competing Interests: The authors declare no competing financial interest.

We have used NMR and computational methods to characterize the dynamics of the ribonuclease barnase over a wide range of timescales in free and inhibitor-bound states. Using temperature- and denaturant-dependent measurements of chemical shift, we show that barnase undergoes frequent and highly populated hinge bending. Using relaxation dispersion, we characterize a slower and less populated motion with a rate of 750 ± 200 s -1 , involving residues around the lip of the active site, which occurs in both free and bound states and therefore suggests conformational selection. Normal mode calculations characterize correlated hinge bending motions on a very rapid timescale. These three measurements are combined with previous measurements and molecular dynamics calculations on barnase to characterize its dynamic landscape on timescales from picoseconds to milliseconds and length scales from 0.1 to 2.5 nm. We show that barnase has two different large-scale fluctuations: one on a timescale of 10 -9 -10 -6 s that has no free energy barrier and is a hinge bending that is determined by the architecture of the protein; and one on a timescale of milliseconds (i.e., 750 s -1 ) that has a significant free energy barrier and starts from a partially hinge-bent conformation. These two motions can be described as hierarchical, in that the more highly populated faster motion provides a platform for the slower (less probable) motion. The implications are discussed. The use of temperature and denaturant is suggested as a simple and general way to characterize motions on the intermediate ns-μs timescale.

Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR-coenzyme complexes. We find that mutagenesis of these second sphere "noncatalytic" residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR-NADH and PETNR-NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy-enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer., Competing Interests: The authors declare no competing financial interest.

Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete 1 H, 15 N and 13 C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the 1 H- 15 N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism.

Talin mediates attachment of the cell to the extracellular matrix. It is targeted by the Rap1 effector RIAM to focal adhesion sites and subsequently undergoes force-induced conformational opening to recruit the actin-interacting protein vinculin. The conformational switch involves the talin R3 domain, which binds RIAM when closed and vinculin when open. Here, we apply pressure to R3 and measure 1 H, 15 N, and 13 C chemical shift changes, which are fitted using a simple model, and indicate that R3 is only 50% closed: the closed form is a four-helix bundle, while in the open state helix 1 is twisted out. Strikingly, a mutant of R3 that binds RIAM with an affinity similar to wild-type but more weakly to vinculin is shown to be 0.84 kJ mol -1 more stable when closed. These results demonstrate that R3 is thermodynamically poised to bind either RIAM or vinculin, and thus constitutes a good mechanosensitive switch., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF 3 - ) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO 3 - ), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the 1 H, 15 N and 13 C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF 3 :ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the 1 H- 15 N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.

Catechol O-methyltransferase (COMT) is an enzyme that plays a major role in catechol neurotransmitter deactivation. Inhibition of COMT can increase neurotransmitter levels, which provides a means of treatment for Parkinson's disease, schizophrenia and depression. COMT exists as two isozymes: a soluble cytoplasmic form (S-COMT), expressed in the liver and kidneys and a membrane-bound form (MB-COMT), found mostly in the brain. Here we report the backbone 1 H, 15 N and 13 C chemical shift assignments of S-COMT in complex with S-adenosyl-L-methionine, 3,5-dinitrocatechol and Mg 2+ . Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97 % of all backbone resonances were assigned in the complex, with 205 out of a possible 215 residues assigned in the 1 H- 15 N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS+ webserver is in good agreement with published X-ray crystal structures.

The effects of sodium thiocyanate, sodium chloride, and sodium sulfate on the ribonuclease barnase were studied using differential scanning calorimetry (DSC) and NMR. Both measurements reveal specific and saturable binding at low anion concentrations (up to 250 mM), which produces localized conformational and energetic effects that are unrelated to the Hofmeister series. The binding of sulfate slows intramolecular motions, as revealed by peak broadening in 13 C heteronuclear single quantum coherence spectroscopy. None of the anions shows significant binding to hydrophobic groups. Above 250 mM, the DSC results are consistent with the expected Hofmeister effects in that the chaotropic anion thiocyanate destabilizes barnase. In this higher concentration range, the anions have approximately linear effects on protein NMR chemical shifts, with no evidence for direct interaction of the anions with the protein surface. We conclude that the effects of the anions on barnase are mediated by solvent interactions. The results are not consistent with the predictions of the preferential interaction, preferential hydration, and excluded volume models commonly used to describe Hofmeister effects. Instead, they suggest that the Hofmeister anion effects on both stability and solubility of barnase are due to the way in which the protein interacts with water molecules, and in particular with water dipoles, which are more ordered around sulfate anions and less ordered around thiocyanate anions., Competing Interests: The authors declare no competing financial interest.

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-D-glucose 1-phosphate (βG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-D-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-D-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.

Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM-peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.

We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N2 state is 97% populated. This allows us to characterize the structure of the "pure" N2 state of ubiquitin. The N2 state shows a substantial change in the orientation of strand β5 compared to that of the normal folded N1 state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion.

It is becoming increasingly clear that proteins transiently populate high-energy excited states as a necessary requirement for function. Here, we demonstrate that rational mutation based on the characteristics of the structure and dynamics of proteins obtained from pressure experiments is a new strategy for amplifying particular fluctuations in proteins. We have previously shown that ubiquitin populates a high-energy conformer, N2, at high pressures. Here, we show that the Q41N mutation favors N2: high-pressure nuclear magnetic resonance (NMR) shows that N2 is ∼70% populated in Q41N but only ∼20% populated in the wild type at ambient pressure. This allows us to characterize the structure of N2, in which α1-helix, the following loop, β3-strand, and β5-strand change their orientations relative to the remaining regions. Conformational fluctuation on the microsecond time scale, characterized by (15)N spin relaxation NMR analysis, is markedly increased for these regions of the mutant. The N2 conformers produced by high pressure and by the Q41N mutation are quite similar in both structure and dynamics. The conformational change to produce N2 is proposed to be a novel dynamic feature beyond the known recognition dynamics of the protein. Indeed, it is orthogonal to that seen when proteins containing a ubiquitin-interacting motif bind at the hydrophobic patch of ubiquitin but matches changes seen on binding to the E2 conjugating enzyme. More generally, structural and dynamic effects of hydrodynamic pressure are shown to be useful for characterizing functionally important intermediates.

Coupling of fast protein dynamics to enzyme chemistry is controversial and has ignited considerable debate, especially over the past 15 years in relation to enzyme-catalyzed H-transfer. H-transfer can occur by quantum tunneling, and the temperature dependence of kinetic isotope effects (KIEs) has emerged as the "gold standard" descriptor of these reactions. The anomalous temperature dependence of KIEs is often rationalized by invoking fast motions to facilitate H-transfer, yet crucially, direct evidence for coupled motions is lacking. The fast motions hypothesis underpinning the temperature dependence of KIEs is based on inference. Here, we have perturbed vibrational motions in pentaerythritol tetranitrate reductase (PETNR) by isotopic substitution where all non-exchangeable atoms were replaced with the corresponding heavy isotope ((13)C, (15)N, and (2)H). The KIE temperature dependence is perturbed by heavy isotope labeling, demonstrating a direct link between (promoting) vibrations in the protein and the observed KIE. Further we show that temperature-independent KIEs do not necessarily rule out a role for fast dynamics coupled to reaction chemistry. We show causality between fast motions and enzyme chemistry and demonstrate how this impacts on experimental KIEs for enzyme reactions.

Experimental observations of fluoromagnesate and fluoroaluminate complexes of β-phosphoglucomutase (β-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of β-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of β-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.

The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.

Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the alpha-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of beta-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO-3 , but is MgF-3 (trifluoromagnesate). Although MgF-3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three beta-PGM-MgF-3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F...H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the beta-PGM-MgF--3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF--3 model of near-transition state conformations.

(19)F-based NMR analysis and hydrogen/deuterium primary isotope shifts establish the formation of a highly populated solution-state trigonal bipyramidal complex involving beta-phosphoglucomutase (beta-PGM), alpha-galactose 1-phosphate (alphaGal1P), and trifluoromagnesate (MgF(3)(-)), PGM-MgF(3)-alphaGal1P, that is a transition state analogue for phosphoryl transfer. Full backbone resonance assignment of the protein shows that its structure is in the closed conformation required for catalytic activity and is closely related to the corresponding complex with glucose 6-phosphate, which we have recently identified using NMR analysis in solution and X-ray crystallography in the solid state. The previous identification of three structural waters in a PGM-alphaGal1P binary substrate complex had indicated that, in the presence of alphaGal1P, magnesium ions, and fluoride, beta-PGM should indeed form a PGM-MgF(3)-alphaGal1P-TSA complex whereas, in the solid-state, apparently it did not. This cast doubt on the validity of the interpretation of MgF(3)(-) complexes. The present work establishes that, in solution, the expectation that a PGM-MgF(3)-alphaGal1P-TSA complex should readily form is fulfilled. These results thus refute the final evidence used to claim that the trigonal bipyramidal species observed in some solid-state structures of complexes involving beta-PGM are pentaoxyphosphorane intermediates.

The isomerization of beta-glucose-1-phosphate (betaG1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (betaPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (betaG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (kcat = 65 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.7 microM, Ki = 122 microM). If alphaG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (kcat = 74 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.8 microM, Ki = 122 microM, K(alphaG16BP) = 91 microM for productive binding, K(alphaG16BP) = 21 microM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140,000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the betaPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a betaG1P x MgF3(-) x betaPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M(5) and suggests an affinity of the MgF3(-) moiety to this transition-state analogue (TSA) of < or = 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of betaPGM.

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. beta-Phosphoglucomutase catalyses the isomerization of beta-glucose-1-phosphate to beta-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using (19)F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF(3)(-) mimics the transferring PO(3)(-) moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of beta-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.

The 2A proteinases (2A(pro)) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2A(pro) also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2A(pro) (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2A(pro) substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2A(pro), but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2A(pro) translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2A(pro), particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s(-1). This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.

The genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Methylococcus capsulatus (Bath) were localised to an 8.3-kb EcoRI fragment of the genome. Genes encoding the large subunit ( cbbL), small subunit ( cbbS) and putative regulatory gene ( cbbQ) were shown to be located on one cluster. Surprisingly, cbbO, a second putative regulatory gene, was not located in the remaining 1.2-kb downstream (3') of cbbQ. However, probing of the M. capsulatus (Bath) genome with cbbO from Nitrosomonas europaea demonstrated that a cbbO homologue was contained within a separate 3.0-kb EcoRI fragment. Instead of a cbbR ORF being located upstream (5') of cbbL, there was a moxR-like ORF that was transcribed in the opposite direction to cbbL. There were three additional ORFs within the large 8.3-kb EcoRI fragment: a pyrE-like ORF, an rnr-like ORF and an incomplete ORF with no sequence similarity to any known protein. Phylogenetic analysis of cbbL from M. capsulatus (Bath) placed it within clade A of the green-type Form 1 Rubisco. cbbL was expressed in M. capsulatus (Bath) when grown with methane as a sole carbon and energy source under both copper-replete and copper-limited conditions. M. capsulatus (Bath) was capable of autotrophic growth on solid medium but not in liquid medium. Preliminarily investigations suggested that other methanotrophs may also be capable of autotrophic growth. Rubisco genes were also identified, by PCR, in Methylococcus-like strains and Methylocaldum species; however, no Rubisco genes were found in Methylomicrobium album BG8, Methylomonas methanica S1, Methylomonas rubra, Methylosinus trichosporium OB3b or Methylocystis parvus OBBP.

Polyphenols are largely responsible for the astringency and "mouthfeel" of tea and wine by their interactions with basic salivary proline-rich proteins. Astringency arises from precipitation of polyphenol/peptide complexes, which is an important protective mechanism in animals that consume polyphenols. This paper presents biophysical studies of the interactions between chemically defined polyphenols and peptides. It is shown that intermolecular binding is dominated by stacking of polyphenolic rings onto planar hydrophobic surfaces and is strengthened by multiple cooperative binding of polyphenolic rings. Affinities weaken at higher temperatures and are unaffected by pH between pH 3.8 and 6.0. Measurements of self-diffusion rates for peptides with increasing concentrations of polyphenol demonstrate that peptides become increasingly coated with polyphenol. When the coating is sufficiently extensive to provide cooperative polyphenol bridges, the peptide dimerizes and precipitates. Light scattering measurements and electron microscopy indicate that the insoluble particles fall into two discrete size classes of ca. 80 and 500 nm diameter. The larger particles are favored at higher temperature and pH, suggesting that the particles are in a colloidal state, with the smaller particles being stabilized by charge repulsion between particles, and that precipitation of the complexes may be a phase separation process.

Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.

The solution structure of an N-terminally truncated and mutant form (M65L(2-98)) of the human cysteine protease inhibitor cystatin A has been reported that reveals extensive structural differences when compared to the previously published structure of full-length wild-type (WT) cystatin A. On the basis of the M65L(2-98) structure, a model of the inhibitory mechanism of cystatin A was proposed wherein specific interactions between the N- and C-terminal regions of cystatin A are invoked as critical determinants of protease binding. To test this model and to account for the reported differences between the two structures, we undertook additional structural and mechanistic analyses of WT and mutant forms of human cystatin A. These show that modification at the C-terminus of cystatin A by the addition of nine amino acids has no effect upon the affinity of papain inhibition (K(D) = 0.18+/-0.02 pM) and the consequences of such modification are not propagated to other parts of the structure. These findings indicate that perturbation of the C-terminus can be achieved without any measurable effect on the N-terminus or the proteinase binding loops. In addition, introduction of the methionine-65 --> leucine substitution into cystatin A that retains the N-terminal methionine (M65L(1-98)) has no significant effect upon papain binding (K(D) = 0.34+/-0.02 pM). Analyses of the structures of WT and M65L(1-98) using (1)H NMR chemical shifts and residual dipolar couplings in a partially aligning medium do not reveal any evidence of significant differences between the two inhibitors. Many of the differences between the published structures correspond to major violations by M65L(2-98) of the WT constraints list, notably in relation to the position of the N-terminal region of the inhibitor, one of three structural motifs indicated by crystallographic studies to be involved in protease binding by cystatins. In the WT structure, and consistent with the crystallographic data, this region is positioned adjacent to another inhibitory motif (the first binding loop), whereas in M65L(2-98) there is no proximity of these two motifs. As the NMR data for both WT9C and M65L(1-98) are wholly consistent with the published structure of WT cystatin A and incompatible with that of M65L(2-98), we conclude that the former represents the most reliable structural model of this protease inhibitor.

Prions, the causative agents of Creutzfeldt-Jacob Disease (CJD) in humans and bovine spongiform encephalopathy (BSE) and scrapie in animals, are principally composed of PrPSc, a conformational isomer of cellular prion protein (PrPC). The propensity of PrPC to adopt alternative folds suggests that there may be an unusually high proportion of alternative conformations in dynamic equilibrium with the native state. However, the rates of hydrogen/deuterium exchange demonstrate that the conformation of human PrPC is not abnormally plastic. The stable core of PrPC has extensive contributions from all three alpha-helices and shows protection factors equal to the equilibrium constant for the major unfolding transition. A residual, hyper-stable region is retained upon unfolding, and exchange analysis identifies this as a small nucleus of approximately 10 residues around the disulfide bond. These results show that the most likely route for the conversion of PrPC to PrPSc is through a highly unfolded state that retains, at most, only this small nucleus of structure, rather than through a highly organized folding intermediate.

It is demonstrated that the identity of residues accessing excited conformational states that are of low free energy relative to the ground state in proteins can be obtained from amide proton NMR chemical shift temperature dependences displaying significant curvature. For the N-terminal domain of phosphoglycerate kinase, hen egg-white lysozyme and BPTI, conformational heterogeneity arises from a number of independent sources, including: structural instability resulting from deletion of part of the protein; a minor conformer generated through disulphide bond isomerisation; an alternative hydrogen bond network associated with buried water molecules; alternative hydrogen bonds involving backbone amides and surface-exposed side-chain hydrogen bond acceptors; and the disruption of loops, ends of secondary structural elements and chain termini. In many of these cases, the conformational heterogeneity at these sites has previously been identified by X-ray and/or NMR studies, but conformational heterogeneity of buried water molecules has hitherto received little attention. These multiple independent low free-energy excited states each involve a small number of residues and are shown to be within 2.5 kcal mol-1 of the ground state. Their relationship with the partially unfolded forms previously characterised using amide proton exchange studies is discussed., (Copyright 1998 Academic Press)

Temperature coefficients have been measured by 2D NMR methods for the amide and C alpha H proton chemical shifts in two globular proteins, bovine pancreatic trypsin inhibitor and hen egg-white lysozyme. The temperature-dependent changes in chemical shift are generally linear up to about 15 degrees below the global denaturation temperature, and the derived coefficients span a range of roughly -16 to +2 ppb/K for amide protons and -4 to +3 ppb/K for C alpha H. The temperature coefficients can be rationalized by the assumption that heating causes increases in thermal motion in the protein. Precise calculations of temperature coefficients derived from protein coordinates are not possible, since chemical shifts are sensitive to small changes in atomic coordinates. Amide temperature coefficients correlate well with the location of hydrogen bonds as determined by crystallography. It is concluded that a combined use of both temperature coefficients and exchange rates produces a far more reliable indicator of hydrogen bonding than either alone. If an amide proton exchanges slowly and has a temperature coefficient more positive than -4.5 ppb/K, it is hydrogen bonded, while if it exchanges rapidly and has a temperature coefficient more negative than -4.5 ppb/K, it is not hydrogen bonded. The previously observed unreliability of temperature coefficients as measures of hydrogen bonding in peptides may arise from losses of peptide secondary structure on heating.

Polyphenols (tannins) in the diet not only precipitate oral proteins, producing an astringent sensation, but also interact with dietary proteins and digestive enzymes in the gut, resulting in a variety of antinutritive and toxic effects. Salivary proline-rich proteins (PRPs), which are secreted into the oral cavity, form complexes with and precipitate dietary polyphenols, and thus, they constitute the primary mammalian defense directed against ingested tannins. In order to characterize the interaction, NMR studies were performed which involved titrating a series of polyphenols into a synthetic 19-residue PRP fragment. The results show that the predominant mode of association is a hydrophobic stacking of the polyphenol ring against the pro-S face of proline and that the first proline residue of a Pro-Pro sequence is a particularly favored binding site. Measurement of dissociation constants indicates that the larger and more complex polyphenols interact more strongly with the PRP fragment; the order of binding affinity was determined as procyanidin dimer B-2 > pentagalloylglucose > trigalloylglucose >> proanthocyanidin monomer (-)-epicatechin approximately propyl gallate. Smaller polyphenols can bind with one phenolic ring stacked against each proline residue, whereas larger polyphenols occupy two or three consecutive prolines. The more complex polyphenols interact with the PRP fragment in a multidentate fashion; moreover, they self-associate or stack when bound. Thus, a model is proposed in which multiple polyphenol/polyphenol and polyphenol/PRP interactions act cooperatively to achieve precipitation.

The protein IB5 has been purified from human parotid saliva. This protein contains several repeats of a short proline-rich sequence. Dissociation constants have been measured at several discrete binding sites using 1H-NMR for the hydrolysable tannins (polyphenols) beta-1,3,6-tri-O-galloyl-D-glucopyranose, beta-1,2,4,6-tetra-O-galloyl-D-glucopyranose and beta-1,2,3,4,6-penta-O-galloyl-D-glucopyranose and the condensed proanthocyanidin (--)-epicatechin. The dissociation constants for trigalloyl glucose and pentagalloyl glucose were 15 X 10(-5) and 1.7 X 10(-5) M, respectively, which are 115 and 1660 times stronger than those previously measured under the same conditions for a single repeat of a mouse salivary proline-rich protein. The increase in affinity is ascribed to intramolecular secondary interactions, which are strengthened by the rigidity of the interacting molecules.