Random Coil Chemical Shifts Research Articles

NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding.

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.

Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form

Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S](2+/1+) cluster and one [3Fe-4S](1+/0) cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys(11)-Xaa-Xaa-Asp(14)-Xaa-Xaa-Cys(17)-Xaa(n)-Cys(51)-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S]<-->[4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg.l(-1)), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl(3) together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.

Random coil chemical shifts in acidic 8 M urea: implementation of random coil shift data in NMRView.

Studies of proteins unfolded in acid or chemical denaturant can help in unraveling events during the earliest phases of protein folding. In order for meaningful comparisons to be made of residual structure in unfolded states, it is necessary to use random coil chemical shifts that are valid for the experimental system under study. We present a set of random coil chemical shifts obtained for model peptides under experimental conditions used in studies of denatured proteins. This new set, together with previously published data sets, has been incorporated into a software interface for NMRView, allowing selection of the random coil data set that fits the experimental conditions best.

Random-coil chemical shifts of phosphorylated amino acids.

The 1H, 13C, 15N and 31P random-coil chemical shifts and phosphate pKa values of phosphorylated amino acids pSer, pThr and pTyr in the protected peptide Ac-Gly-Gly-X-Gly-Gly-NH2 have been obtained in water at 25 degrees C over the pH range 2 to 9. Analysis of ROESY spectra indicates that the peptides are unstructured. Phosphorylation induces changes in random-coil chemical shifts, some of which are comparable to those caused by secondary structure formation, and are therefore significant in structural analyses based on the chemical shift.

Use of 13C conformation-dependent chemical shifts to elucidate the local structure of a large protein with homologous domains in solution and solid state

In order to clarify the difference between solution NMR and X-ray diffraction analyses concerning the presence of α-helical structure in protein A, the 13C conformation-dependent chemical shifts of the 13C-labeled carbonyl carbons for selectively labeled protein A were used. In the 13C CP/MAS NMR spectra, the higher-field shifts of the carbonyl carbons of 13C-labeled Thr and Val residues compared with the random coil chemical shifts both in solution and solid state imply the presence of the third helix in the polypeptide chain, in contrast to the crystal structure of Fc-bound B-domain. Thus, a combination of selective isotope labeling and conformation-dependent chemical shifts will be a good Indicator to monitor the local structure of homologous protein in solution and solid state.

NMR spectroscopic studies of the DNA-binding domain of the monomer-binding nuclear orphan receptor, human estrogen related receptor-2. The carboxyl-terminal extension to the zinc-finger region is unstructured in the free form of the protein.

Unlike steroid and retinoid receptors, which associate with DNA as dimers, human estrogen related receptor-2 (hERR2) belongs to a growing subclass of nuclear hormone receptors that bind DNA with high affinity as monomers. A carboxyl-terminal extension (CTE) to the zinc-finger domain has been implicated to be responsible for determining the stoichiometry of binding by a nuclear receptor to its response element. To better understand the mechanism by which DNA specificity is achieved, the solution structure of the DNA-binding domain of hERR2 (residues 96-194) consisting of the two putative zinc fingers and the requisite 26-amino acid CTE was analyzed by multidimensional heteronuclear magnetic resonance spectroscopy. The highly conserved zinc-finger region (residues 103-168) has a fold similar to those reported for steroid and retinoid receptors, with two helices that originate from the carboxyl-terminal ends of the two zinc fingers and that pack together orthogonally, forming a hydrophobic core. The CTE element of hERR2 is unstructured and highly flexible, exhibiting nearly random coil chemical shifts, extreme sensitivity of the backbone amide protons to solvent presaturation, and reduced heteronuclear (1H-15N) nuclear Overhauser effect values. This is in contrast to the dimer-binding retinoid X and thyroid hormone receptors, where, in each case, a helix has been observed within the CTE. The implications of this property of the hERR2 CTE are discussed.

Precise Limits of the N-Terminal Domain of DnaB Helicase Determined by NMR Spectroscopy

Two separate N-terminal fragments of the 470-amino-acidEscherichia coliDnaB helicase, comprising residues 1-142 and 1-161, were expressed inE. coli.The proteins were extracted in a soluble fraction, purified, and characterised physically. In contrast to the full-length protein, which is hexameric, both fragments exist as monomers in solution, as demonstrated by sedimentation equilibrium measurements. CD spectroscopy was used to confirm that the 161-residue fragment is highly structured (mostly α-helical) and undergoes reversible thermal denaturation. The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by assignment of resonances from flexible residues in NMR spectra. The1H NMR signals of the flexible residues are located at random coil chemical shifts, and their linewidths are significantly narrower than those of the structured core, indicating complete disorder and increased mobility on the nanosecond time scale. The results support the idea of a flexible hinge region between the N- and C-terminal domains of the native hexameric DnaB protein.

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects

In this study we report on the 1H, 13C and 15N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of 1H, 13C and 15N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.

Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state.

The structures of the major folding intermediate, the transition state for folding, and the folded state of barnase have been previously characterized. We now add a further step toward a complete picture of the folding of barnase by reporting the backbone 15N, 13C, and 1H NMR assignments for barnase unfolded at pH 1.8 and 30 degrees C. These assignments, which were obtained from a combination of heteronuclear magnetization transfer and backbone triple-resonance NMR experiments, constitute the first stage in the structural characterization of this denatured state by NMR. Interresidue nuclear Overhauser effect contacts and deviations from 1H random-coil chemical shifts provide evidence for stable residual structure. The structured regions span residues in the native protein that contain its major alpha-helix and central strands of the beta-sheet. Earlier experiments have shown that these regions are predominantly intact in the major folding intermediate and that their docking is partly rate determining in folding.

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.

Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using proton NMR

We have overproduced and purified the carboxy-terminal transactivation domain of Vmw65 (VP16) of herpes simplex virus, and studied potential folding of the domain by 1H NMR. Two species of the acidic domain were obtained from the bacterial expression system, and we demonstrate that one of these represents read-through of the natural amber termination codon of the Vmw65 reading frame producing a larger polypeptide. Additional residues in the read-through product were identified by total amino acid analysis and by NMR. Study of the correctly terminated product by 1D NMR gave resonances which were clustered into groups around their random-coil chemical shift positions, and 2D NMR demonstrated that, even in mixed solvents containing up to 80% MeOH, there was very little evidence of secondary structure. Together these results indicate that the isolated acid domain has little if any alpha-helical content of any stable nature. We discuss these results with reference to the demonstrated activity of the acidic domain in a wide variety of polypeptide contexts.