Conformational Exchange Processes Research Articles

Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

NMR spin relaxation in the rotating frame (R(1rho)) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide 1H off-resonance R(1rho) relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using 15N or 13C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The 1H off-resonance R(1rho) data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 micros. This is in contrast to the case for 15N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly 15N-enriched samples that were either highly enriched in 2H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the 1H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present 1H off-resonance R(1rho) experiments are in excellent agreement with those determined previously using a combination of 15N laboratory-frame and off-resonance R(1rho) relaxation data, with average values of [see text] and 21 +/- 3 micros , respectively.

Gated Electron Transfers and Electron Pathways in Azurin: A NMR Dynamic Study at Multiple Fields and Temperatures

Dynamic properties of electron transfer pathways in a small blue copper cupredoxin are explored using an extensive 15N NMR relaxation study of reduced Pseudomonas aeruginosa azurin at four magnetic fields (500-900 MHz) and at two temperatures chosen well below the melting point of the protein. Following a careful model-free analysis, several protein regions with different dynamic regimes are identified. Nanosecond time-scale mobility characterizes various residues of the hydrophobic surface patch believed to mark the natural entry point for electrons, notably the surface-exposed copper-ligand His117. These findings are consistent with a gated electron transfer process according to the "dynamic docking" model. Residues 47-49 along intramolecular pathways of electrons show rigidity that is remarkably conserved when increasing the temperature. Three different conformational exchange processes were observed in the millisecond range, one near the only disulfide bridge in the molecule and two near the copper ion. The latter two processes are consistent with previous data such as crystal structures at various pH values and NMR relaxation dispersion experiments; they may indicate an additional gated electron transfer mechanism at slower time-scales.

The integral membrane enzyme PagP alternates between two dynamically distinct states.

PhoPQ-activated gene P (PagP) is an integral membrane enzyme that transfers the sn-1 palmitate chain from phospholipid to lipopolysaccharide in Gram-negative bacteria. A recent x-ray crystallographic study established that the sn-1 palmitate binds within a long cavity at the center of the PagP beta barrel. The high mobility required to permit substrate entry into the central core of the barrel contrasts with the need to assemble a well defined structure in the peripheral loops, where many key catalytic residues are located. To gain insight into how dynamics relate to the function of PagP, the enzyme was reconstituted into CYFOS-7, a detergent that supports enzymatic activity. Under these conditions, PagP exists in equilibrium between two states, relaxed (R) and tense (T). The kinetics and thermodynamics of the interchange have been investigated by (1)H-(15)N NMR spectroscopy, with Delta H = -10.7 kcal/mol and Delta S = -37.5 cal/mol.K for the R--> T transition. A comparison of chemical shifts between the two states indicates that major structural changes occur in the large extracellular L1 loop and adjacent regions of the beta barrel. In addition to the R,T interconversion, other conformational exchange processes are observed in the R state, showing it to be quite flexible. Thus a picture emerges in which substrate entry is facilitated by the mobility of the R state, whereas the relatively rigid T state adopts a radically different conformation in a region of the protein known to be essential for catalysis. The ability to switch between dynamically distinct states may be a key feature of the catalytic cycle of PagP.

Peptaibol Zervamicin IIB Structure and Dynamics Refinement from Transhydrogen Bond J Couplings

Zervamicin IIB (Zrv-IIB) is a channel-forming peptaibol antibiotic of fungal origin. The measured transhydrogen bond 3h J NC′ couplings in methanol solution heaving average value of −0.41 Hz indicate that the stability of the Zrv-IIB helix in this milieu is comparable to the stability of helices in globular proteins. The N-terminus of the peptide forms an α-helix, whereas 3 10-helical hydrogen bonds stabilize the C-terminus. However, two weak transhydrogen bond peaks are observed in a long-range HNCO spectrum for HN Aib 12. Energy calculations using the Empirical Conformation Energy Program for Peptides (ECEPP)/2 force field and the implicit solvent model show that the middle of the peptide helix accommodates a bifurcated hydrogen bond that is simultaneously formed between HN Aib 12 and CO Leu 8 and CO Aib 9. Several lowered 3h J NC′ on a polar face of the helix correlate with the conformational exchange process observed earlier and imply dynamic distortions of a hydrogen bond pattern with the predominant population of a properly folded helical structure. The refined structure of Zrv-IIB on the basis of the observed hydrogen bond pattern has a small (∼20°) angle of helix bending that is virtually identical to the angle of bending in dodecylphosphocholine (DPC) micelles, indicating the stability of a hinge region in different environments. NMR parameters ( 1HN chemical shifts and transpeptide bond 1 J NC′ couplings) sensitive to hydrogen bonding along with the solvent accessible surface area of carbonyl oxygens indicate a large polar patch on the convex side of the helix formed by three exposed backbone carbonyls of Aib 7, Aib 9, and Hyp 10 and polar side chains of Hyp 10, Gln 11, and Hyp 13. The unique structural features, high helix stability and the enhanced polar patch, set apart Zrv-IIB from other peptaibols (for example, alamethicin) and possibly underlie its biological and physiological properties.

Characterization of micros-ms dynamics of proteins using a combined analysis of 15N NMR relaxation and chemical shift: conformational exchange in plastocyanin induced by histidine protonations.

An approach is presented that allows a detailed, quantitative characterization of conformational exchange processes in proteins on the micros-ms time scale. The approach relies on a combined analysis of NMR relaxation rates and chemical shift changes and requires that the chemical shift of the exchanging species can be determined independently of the relaxation rates. The applicability of the approach is demonstrated by a detailed analysis of the conformational exchange processes previously observed in the reduced form of the blue copper protein, plastocyanin from the cyanobacteria Anabaena variabilis (A.v. PCu) (Ma, L.; Hass, M. A. S.; Vierick, N.; Kristensen, S. M.; Ulstrup, J.; Led, J. J. Biochemistry 2003, 42, 320-330). The R1 and R2 relaxation rates of the backbone 15N nuclei were measured at a series of pH and temperatures on an 15N labeled sample of A.v. PCu, and the 15N chemical shifts were obtained from a series of HSQC spectra recorded in the pH range from 4 to 8. From the R1 and R2 relaxation rates, the contribution, Rex, to the transverse relaxation caused by the exchanges between the different allo-states of the protein were determined. Specifically, it is demonstrated that accurate Rex terms can be obtained from the R1 and R2 rates alone in the case of relatively rigid proteins with a small rotational anisotropy. The Rex terms belonging to the same exchange process were identified on the basis of their pH dependences. Subsequently the identifications were confirmed quantitatively by the correlation between the Rex terms and the corresponding chemical shift differences of the exchanging species. By this approach, the Rex terms of 15N nuclei belonging to contiguous regions in the protein could be assigned to the same exchange process. Furthermore, the analysis of the exchange terms shows that the observed micros-ms dynamics in A.v. PCu are caused primarily by the protonation/deprotonation of two histidine residues, His92 and His61, His92 being ligated to the Cu(I) ion. Also the exchange rate of the protonation/deprotonation process of His92 and its pH and temperature dependences were determined, revealing a reaction pathway that is more complex than a simple specific-acid/base catalysis. Finally, the approach allows a differentiation between two-site and multiple-site exchange processes, thus revealing that the protonation/deprotonation of His61 is at least a three-site exchange process. Overall, the approach makes it feasible to obtain exchange rates that are sufficiently accurate and versatile for studies of the kinetics and the mechanisms of local protein dynamics on the sub-millisecond time scale.

Quantitative analysis of conformational exchange contributions to 1H-15N multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant.

Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential 1H-15N multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 +/- 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the 1H and 15N chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential 1H-15N multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from 15N off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the 1H and 15N spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the 1H chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on 1H single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization

The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme β-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid–water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni–NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni–NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective 15N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial 15N, 1H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.

Identification of a Residue Critical for Maintaining the Functional Conformation of BPTI

The effects of amino acid replacements on the backbone dynamics of bovine pancreatic trypsin inhibitor (BPTI) were examined using 15N NMR relaxation experiments. Previous studies have shown that backbone amide groups within the trypsin-binding region of the wild-type protein undergo conformational exchange processes on the μs time scale, and that replacement of Tyr35 with Gly greatly increases the number of backbone atoms involved in such motions. In order to determine whether these mutational effects are specific to the replacement of this residue with Gly, six additional replacements were examined in the present study. In two of these, Tyr35 was replaced with either Ala or Leu, and the other four were single replacements of Tyr23, Phe33, Asn43 or Asn44, all of which are highly buried in the native structure and conserved in homologous proteins. The Y35A and Y35L mutants displayed dynamic properties very similar to those of the Y35G mutant, with the backbone segments including residues 10–19 and 32–44 undergoing motions revealed by enhanced 15N transverse relaxation rates. On the other hand, the Y23L, N43G and N44A substitutions caused almost no detectable changes in backbone dynamics, on either the ns–ps or ms–μs time scales, even though each of these replacements significantly destabilizes the native conformation. Replacement of Phe33 with Leu caused intermediate effects, with several residues that have previously been implicated in motions in the wild-type protein displaying enhanced transverse relaxation rates. These results demonstrate that destabilizing amino acid replacements can be accommodated in a native protein with dramatically different effects on conformational dynamics and that Tyr35 plays a particularly important role in defining the conformation of the trypsin-binding site of BPTI.

Off-resonance TROSY (R1 rho - R1) for quantitation of fast exchange processes in large proteins.

Current solution NMR experiments for characterizing conformational exchange processes in large proteins are limited to exchange rates ca. 500-3000 s-1. A TROSY-based constant relaxation time (R1rho - R1) experiment is designed to extend this capability to measure motion with rates up to 105 s-1 in large macromolecules. The experiment combines off-resonance spin-lock rf fields, which provide access to the faster time-scale dynamics, with TROSY coherence selection, which extends the molecular-weight range available for study. When implemented on the 53-kDa dimeric enzyme triosephosphate isomerase, the experiment yielded substantial gains in signal-to-noise (up to 60%) over current experiments at modest static magnetic fields (14.1 T). The TROSY (R1rho - R1) experiment should therefore be of general utility for investigation of fast conformational exchange events in large proteins.

Structure and dynamics of copper-free SOD: The protein before binding copper.

The solution structure of the copper-free state of a monomeric form of superoxide dismutase (153 amino acids) was determined through (13)C and (15)N labeling. The protein contained two mutations at the native subunit-subunit interface (F50E and G51E) to obtain a soluble monomeric species and a mutation in the active site channel (E133Q). About 93% of carbon atoms, 95% of nitrogen atoms, and 96% of the protons were assigned. A total of 2467 meaningful NOEs and 170 dihedral angles provided a family of 35 conformers with RMSD values of 0.76 +/- 0.09 A for the backbone and 1.22 +/- 0.13 A for all heavy atoms. The secondary structure elements, connected by loops, produce the typical superoxide dismutase Greek key fold, formed by an eight-stranded beta-barrel. The comparison with the copper-bound monomeric and dimeric structures shows that the metal ligands have a conformation very close to that of the copper-bound forms. This feature indicates that the copper-binding site is preorganized and well ordered also in the absence of the copper ion. The active-site channel shows a sizable increase in width, achieving a suitable conformation to receive the copper ion. The histidines ring NH resonances that bind the copper ion and the region around the active-site channel experience, as found from (15)N relaxation studies, conformational exchange processes. The increased width of the channel and the higher mobility of the histidine rings of the copper site in the copper-free form with respect to the holoprotein is discussed in terms of the process of copper insertion.

Solution Structure and Dynamics of the Lipoic Acid-bearing Domain of Human Mitochondrial Branched-chain α-Keto Acid Dehydrogenase Complex

The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain alpha-keto acid dehydrogenase complex plays a central role in substrate channeling in this mitochondrial multienzyme complex. We have employed multidimensional heteronuclear NMR techniques to determine the structure and dynamics of the LBD of the human branched-chain alpha-keto acid dehydrogenase complex (hbLBD). Similar to LBD from other members of the alpha-keto acid dehydrogenase family, the solution structure of hbLBD is a flattened beta-barrel formed by two four-stranded antiparallel beta-sheets. The lipoyl Lys(44) residue resides at the tip of a beta-hairpin comprising a sharp type I beta-turn and the two connecting beta-strands 4 and 5. A prominent V-shaped groove formed by a surface loop, L1, connecting beta 1- and beta 2-strands and the lipoyl lysine beta-hairpin constitutes the functional pocket. We further applied reduced spectral density functions formalism to extract dynamic information of hbLBD from (15)N-T(1), (15)N-T(2), and ((1)H-(15)N) nuclear Overhauser effect data obtained at 600 MHz. The results showed that residues surrounding the lipoyl lysine region comprising the L1 loop and the Lys(44) beta-turn are highly flexible, whereas beta-sheet S1 appears to display a slow conformational exchange process.

Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P. magnus strain ALB8 (ALB8-GA) exhibit 59 % sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The 15N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P. magnus with the ALB8-GA module has been isolated only from humans.

A novel view of domain flexibility in E. coli adenylate kinase based on structural mode-coupling 15N NMR relaxation

Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd and LID, catalyzes the reaction AMP+ATP→2ADP. In the ligand-free enzyme the domains AMPbd and LID execute large-amplitude movements controlling substrate binding and product release during catalysis. Domain flexibility is investigated herein with the slowly relaxing local structure (SRLS) model for 15N relaxation. SRLS accounts rigorously for coupling between the global and local N-H motions through a local ordering potential exerted by the protein structure at the N-H bond. The latter reorients with respect to its protein surroundings, which reorient on the slower time scale associated with the global protein tumbling. AKeco diffuses globally with correlation time τm=15.1 ns, while locally two different dynamic cases prevail. The domain CORE features ordering about the equilibrium N-H bond orientation with order parameters, S2, of 0.8-0.9 and local motional correlation times, τ, mainly between 5-130 ps. This represents a conventional rigid protein structure with rapid small-amplitude N-H fluctuations. The domains AMPbd and LID feature small parallel (ZM) ordering of S2=0.2-0.5 which can be reinterpreted as high perpendicular (YM) ordering. M denotes the local ordering/local diffusion frame. Local motion about ZM is given by τ∥≈5 ps and local motion of the effective ZM axis about YM by τ⊥=6-11 ns. ZM is tilted at approximately 20° from the N-H bond. The orientation of the YM axis may be considered parallel to the Ci−1α-Ciα axis. The τ⊥ mode reflects collective nanosecond peptide-plane motions, interpretable as domain motion. A powerful new model of protein flexibility/domain motion has been established. Conformational exchange (Rex) processes accompany the τ⊥ mode. The SRLS analysis is compared with the conventional model-free analysis.

Slow dynamics in folded and unfolded states of an SH3 domain.

(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.

NMR evidence for mechanical coupling of phosphate B I-B II transitions with deoxyribose conformational exchange in DNA

The conformational exchange of the phosphate and deoxyribose groups of the DNA oligomers d(GCGTACGC) 2 and d(CGCTAGCG) 2 have been investigated using a combination of homonuclear and heteronuclear NMR techniques. Two-state exchange between phosphate B I and B II conformations and deoxyribose N and S conformations was expressed as percent population of the major conformer, %B I or %S. Sequence context-dependent variations in %B I and %S were observed. The positions of the phosphate and deoxyribose equilibria provide a quantitative measure of the ps to ns timescale dynamic exchange processes in the DNA backbone. Linear correlations between %B I, %S, and previously calculated model free 13C order parameters ( S 2) were observed. The %B I of the phosphates were found to be correlated to the S 2 of the flanking C3′ and C4′ atoms. The %B I was also found to be correlated with the %S and C1′ S 2 of the deoxyribose ring 5′ of the phosphates. The %B I of opposing phosphates is correlated, while the %B I of sequential phosphates is anti-correlated. These correlations suggest that conformational exchange processes in DNA are coupled to each other and are modulated by DNA base sequence, which may have important implications for DNA-protein interactions.

Solution structure and backbone dynamics of an omega-conotoxin precursor.

Nuclear magnetic resonance spectroscopy was used to characterize the solution structure and backbone dynamics of a putative precursor form of omega-conotoxin MVIIA, a 25-amino-acid residue peptide antagonist of voltage-gated Ca(2+) channels. The mature peptide is found in the venom of a fish-hunting marine snail Conus magus and contains an amidated carboxyl terminus that is generated by oxidative cleavage of a Gly residue. The form examined in this study is identical to the mature peptide except for the presence of the unmodified carboxy-terminal Gly. This form, referred to as omega-MVIIA-Gly, has previously been shown to refold and form its disulfides more efficiently than the mature form, suggesting that the presence of the terminal Gly may favor folding in vivo. The nuclear magnetic resonance (NMR) structure determination indicated that the fold of omega-MVIIA-Gly is very similar to that previously determined for the mature form, but revealed that the terminal Gly residue participates in a network of hydrogen bonds involving both backbone and side chain atoms, very likely accounting for the enhanced stability and folding efficiency. (15)N relaxation experiments indicated that the backbone is well ordered on the nanosecond time scale but that residues 9-15 undergo a conformational exchange processes with a time constant of approximately 35 microseconds. Other studies have implicated this segment in the binding of the peptide to its physiological target, and the observed motions may play a role in allowing the peptide to enter the binding site

An improved method for distinguishing between anisotropic tumbling and chemical exchange in analysis of 15N relaxation parameters.

Although an accurate description of global tumbling of a protein is essential for correct analysis of internal motions. proper distinction between the effects of anisotropic rotational diffusion and conformational exchange has remained a challenge. We present a novel two-part filtering procedure designed specifically to distinguish between the effects of anisotropy and conformational exchange. The efficacy of this method is assessed using synthetic data sets. The method is then applied to two proteins of dramatically different size and shape, OspA and SH3. The large size and extreme anisotropy of OspA provide a challenging case, where conformational exchange is a small perturbation of the effects of anisotropy on transverse relaxation rates. Conversely, in the chicken c-Src SH3 domain, with its small size and nearly spherical shape, anisotropy is a small perturbation of the effects of conformational exchange on transverse relaxation rates. Accurate extraction of the global tumbling parameters for each protein allows optimal characterization of conformational exchange processes, as well as ps-ns time scale motions.

Aromatic ring-flipping in supercooled water: implications for NMR-based structural biology of proteins.

We have characterized, for the first time, motional modes of a protein dissolved in supercooled water: the flipping kinetics of phenylalanyl and tyrosinyl rings of the 6 kDa protein BPTI have been investigated by NMR at temperatures between -3 and -16.5 degrees C. At T = -15 degrees C, the ring-flipping rate constants of Tyr 23, Tyr 35, and Phe 45 are smaller than 2 s(-1), i.e., flip-broadening of aromatic NMR lines is reduced beyond detection and averaging of NOEs through ring-flipping is abolished. This allows neat detection of distinct NOE sets for the individual aromatic (1)H spins. In contrast, the rings of Phe 4, Tyr 10, Tyr 21, Phe 22, and Phe 33 are flipping rapidly on the chemical shift time scale with rate constants being in the range from approximately 10(2) to 10(5) s(-1) even at T = -15 degrees C. Line width measurements in 2D [(1)H,(1)H]-NOESY showed that flipping of the Phe 4 and Phe 33 rings is, however, slowed to an extent that the onset of associated line broadening in the fast exchange limit is registered. The reduced ring-flipping rate constant of Phe 45 in supercooled water allowed very precise determination of Eyring activation enthalpy and entropy from cross relaxation suppressed 2D [(1)H,(1)H]-exchange spectroscopy. This yielded DeltaH = 14 +/- 0.5 kcal.mol(-1) and DeltaS = -4 +/- 1 cal.mol(-1).K(-1), i.e., values close to those previously derived by Wagner and Wüthrich for the temperature range from 4 to 72 degrees C (DeltaH = 16 +/- 1 kcal.mol(-1) and DeltaS = 6 +/- 2 cal.mol(-1).K(-1)). The preservation of the so far uniquely low value for DeltaS indicates that the distribution of internal motional modes associated with the ring flip of Phe 45 is hardly affected by lowering T well below 0 degrees C. Hence, if a globular protein does not cold denature, aromatic flipping rates, and thus likely also the rates of other conformational and/or chemical exchange processes occurring in supercooled water, can be expected to be well estimated from activation parameters obtained at ambient T. This is of keen interest to predict the impact of supercooling for future studies of biological macromolecules, and shows that our approach enables one to conduct NMR-based structural biology at below 0 degrees C in an unperturbed aqueous environment. A search of the BioMagResBank indicated that the overwhelming majority of the Phe and Tyr rings (>95%) are flipping rapidly on the chemical shift time scale at ambient T, while our data for BPTI and activation parameters available for ring-flipping in Iso-2-cytochrome c reveal that in these smaller proteins a total of six out of seventeen rings ( approximately 35%) are "frozen in" at T = -15 degrees C. This suggests that a large fraction of Tyr and Phe rings in globular proteins that are flipping rapidly on the chemical shift time scale at ambient T can be effectively slowed in supercooled water. The present investigation demonstrates that supercooling of protein solutions appears to be an effective means to (i) harvest potential benefits of stalled ring-flipping for refining NMR solution structures, (ii) recruit additional aromatic rings for investigating protein dynamics, and (iii) use multiple slowly flipping rings to probe cold denaturation. The implications for NMR-based structural biology in supercooled water are addressed.

Probing the Nature of the Blue-Shifted Intermediate of Photoactive Yellow Protein in Solution by NMR: Hydrogen−Deuterium Exchange Data and pH Studies

The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded. Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data. It is likely that in the crystalline environment the ordered form of pB is strongly favored.

Backbone dynamics of the C-terminal SH2 domain of the p85α subunit of phosphoinositide 3-kinase: effect of phosphotyrosine-peptide binding and characterization of slow conformational exchange processes

The backbone dynamics of the C-terminal SH2 domain from the regulatory subunit p85α (p85α C-SH2) of phosphoinositide 3-kinase has been investigated in the absence of, and in complex with, a high-affinity phosphotyrosine-containing peptide ligand derived from the platelet-derived growth-factor receptor. 15N R1 and R2 relaxation rates and steady-state [1H]-15N NOE values were measured by means of 1H-15N correlated two-dimensional methods and were analyzed within the framework of the model-free formalism. Several residues in the BC loop and in the neighbouring secondary structural elements display fast local dynamics in the absence of phosphotyrosine peptide ligand as evidenced by below-average [1H]-15N NOE values. Furthermore, residue Gln41 (BC3) displays conformational exchange phenomena as indicated by an above-average R2 relaxation rate. Upon binding of the phosphotyrosine peptide, the NOE values increase to values observed for regular secondary structure and the exchange contribution to the R2 relaxation rate for Gln41 (BC3) vanishes. These observations indicate a loss of backbone flexibility upon ligand binding. Substantial exchange contributions for His56 (βD4) and Cys57 (βD5), which are known to make important interactions with the ligand, are attenuated upon ligand binding. Several residues in the βD′-FB region and the BG loop, which contribute to the ligand binding surface of the protein, exhibit exchange terms which are reduced or vanish when the ligand is bound. Together, these observations suggest that ligand binding is accompanied by a loss of conformational flexibility on the ligand binding face of the protein. However, comparison with other SH2 domains reveals an apparent lack of consensus in the changes in dynamics induced by ligand binding. Exchange rates for individual residues were quantified in peptide-complexed p85α C-SH2 from the dependence of the exchange contributions on the CPMG delay in an R2 series and show that peptide-complexed p85α C-SH2 is affected by multiple conformational exchange processes with exchange rate constants from 102 s−1 to 7·103 s−1. Mapping of the exchange-rate constants on the protein surface show a clustering of residues with similar exchange-rate constants and suggests that clustered residues are affected by a common predominant exchange process.