Solution structure of a peptide model of a region important for the folding of alpha-lactalbumin provides evidence for the formation of nonnative structure in the denatured state

Elucidating the properties of the denatured state of proteins under conditions relevant for their folding is a key factor in understanding the folding process. We show that a peptide corresponding to residues 111-120 of human alpha-lactalbumin has a pronounced propensity to adopt nonnative structure in aqueous solution. Two-dimensional NMR provides evidence for a structured, nonnative conformation in fast exchange with a random coil ensemble. A total of 78 Rotating Frame Overhauser Effects (ROEs) were used to calculate the conformation of the structured population. A nonnative cluster of hydrophobic residues involving the side chains of K114, W118, Ll119, and A120 is observed, which helps to stabilize a turn-like conformation in the vicinity of residues 115-118. The structure in 30% (vol/vol) TFE was also calculated. Interestingly, the addition of TFE did not simply amplify the population of the structured conformer observed in H2O, but instead induced a new conformation. The implications for the folding of the intact protein are discussed. We also discuss the implications of this study for the relevance of the use of mixed TFE/H2O solvent systems to study isolated peptides.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Local and long-range interactions in the molten globule state: A study of chimeric proteins of bovine and human alpha-lactalbumin

The molten globule state of alpha-lactalbumin has ordered secondary structure in the alpha-domain, which comprises residues 1 to 34 and 86 to 123. In order to investigate which part of a polypeptide is important for stabilizing the molten globule state of alpha-lactalbumin, we have produced and studied three chimeric proteins of bovine and human alpha-lactalbumin. The stability of the molten globule state formed by domain-exchanged alpha-lactalbumin, in which the amino acid sequence in the alpha-domain comes from human alpha-lactalbumin and that in the beta-domain comes from bovine alpha-lactalbumin, is the same as that of human alpha-lactalbumin and is substantially greater than that of bovine alpha-lactalbumin. Therefore, our results show that the stability of the molten globule state of alpha-lactalbumin is determined by the alpha-domain and the beta-domain is not important for stabilizing the molten globule state. The substitution of residues 1 to 34 of bovine alpha-lactalbumin with those of human alpha-lactalbumin substantially increases the stability of the molten globule state, while the substitution of residues 86 to 123 of bovine alpha-lactalbumin with those of human alpha-lactalbumin decreases the stability of the molten globule state. Therefore, residues 1 to 34 in human alpha-lactalbumin is more important for the stability of the human alpha-lactalbumin molten globule state than residues 86 to 123. The stabilization of the molten globule state due to substitution of both residues 1 to 34 and 86 to 123 is not identical with the sum of the two individual substitutions, demonstrating the non-additivity of the stabilization of the molten globule state. This result indicates that there is a long-range interaction between residues 1 to 34 and 86 to 123 in the molten globule state of human alpha-lactalbumin. The differences in the stabilities of the molten globule states are well correlated with the averaged helical propensity values in the alpha-domain when the long-range interactions are negligible, suggesting that the local interaction is the dominant term for determining the stability of the molten globule state. Our results also indicate that the apparent cooperativity is closely linked to the stability of the molten globule state, even if the molten globule state is weakly cooperative.     

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(111) disulfide.     

NMR characterization of residual structure in the denatured state of protein L

Triple-resonance NMR experiments were used to assign the (13)C(alpha), (13)C(beta), (15)N and NH resonances for all the residues in the denatured state of a destabilized protein L variant in 2 M guanidine. The chemical shifts of most resonances were very close to their random coil values. Significant deviations were observed for G22, L38 and K39; increasing the denaturant concentration shifted the chemical shifts of these residues towards theory random coil values. Medium-range nuclear Overhauser enhancements were detected in segments corresponding to the turn between the first two strands, the end of the second strand through the turn between the second strand and the helix, and the turn between the helix and the third strand in 3D H(1), N(15)-HSQC-NOESY-HSQC experiments on perdeuterated samples. Longer-range interactions were probed by measuring the paramagnetic relaxation enhancement produced by nitroxide spin labels introduced via cysteine residues at five sites around the molecule. Damped oscillations in the magnitude of the paramagnetic relaxation enhancement as a function of distance along the sequence suggested native-like chain reversals in the same three turn regions. The more extensive interactions within the region corresponding to the first beta-turn than in the region corresponding to the second beta-turn suggests that the asymmetry in the folding reaction evident in previous studies of the protein L folding transition state is already established in the denatured state.     

Diketopiperazine-mediated peptide formation in aqueous solution II. Catalytic effect of phosphate

The previous paper (I) reported that DKP (glycine anhydride) spontaneously reacts with glycine (Gly) or oligoglycines (Gly _n ) to produce longer oligoglycines (Gly _n+2). This paper presents that phosphate catalyzes the condensation reaction quite effectively.Formation of Gly₄ from DKP (0.1 M) and Gly₂ (0.1 M) in phosphate solution of various concentrations was investigated at a neutral pH at 41 °C. The yields of Gly₄ increased almost linearly with the concentration of phosphate from 0.06 M to 0.24 M. The yield in 0.24 M phosphate solution was approximately one hundred times as high as that in the absence of the phosphate, whereas in the case of Gly₃ formation from DKP and Gly the effect of the phosphate was of ten times lower than in the former case. Orthophosphate was the most effective catalyst among the various kind of chemicals tried in the present investigation including polyphosphates.     

NMR solution structure of human cannabinoid receptor-1 helix 7/8 peptide: Candidate electrostatic interactions and microdomain formation

We report the NMR solution structure of a synthetic 40-mer (T³⁷⁷-E⁴¹⁶) that encompasses human cannabinoid receptor-1 (hCB1) transmembrane helix 7 (TMH7) and helix 8 (H8) [hCB1(TMH7/H8)] in 30% trifluoroethanol/H₂O. Structural features include, from the peptide’s amino terminus, a hydrophobic α-helix (TMH7); a loop-like, 11 residue segment featuring a pronounced Pro-kink within the conserved NPxxY motif; a short amphipathic α-helix (H8) orthogonal to TMH7 with cationic and hydrophobic amino-acid clusters; and an unstructured C-terminal end. The hCB1(TMH7/H8) NMR solution structure suggests multiple electrostatic amino-acid interactions, including an intrahelical H8 salt bridge and a hydrogen-bond network involving the peptide’s loop-like region. Potential cation-π and cation-phenolic OH interactions between Y³⁹⁷ in the TMH7 NPxxY motif and R⁴⁰⁵ in H8 are identified as candidate structural forces promoting interhelical microdomain formation. This microdomain may function as a flexible molecular hinge during ligand-induced hCB1 conformer transitions.     

Energetic basis of structural stability in the molten globule state: alpha-lactalbumin

The denatured states of alpha-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of alpha-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of alpha-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of alpha-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.     

A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease.

Hydrogen exchange kinetics were measured on the native states of wild type staphylococcal nuclease and four mutants with values of mGuHCl (defined as dDeltaG/d[guanidine hydrochloride]) ranging from 0.8 to 1.4 of the wild type value. Residues within the five-strand beta-barrel of wild type and E75A and D77A, two mutants with reduced values of m GuHCl, were significantly more protected from exchange than expected on the basis of global stability as measured by fluorescence. In contrast, mutants V23A and M26G with elevated values of mGuHCl approach a flat profile of more or less constant protection independent of position in the structure. Differences in exchange protection between the C-terminus and the beta-barrel region correlate with mGuHCl, suggesting that a residual barrel-like structure becomes more highly populated in the denatured states of m- mutants and less populated in m+ mutants. Variations in the population of such a molten globule-like structure would account for the large changes in solvent accessible surface area of the denatured state thought to underlie m value effects.     

Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.     

Structure characterization of human serum proteins in solution and dry state.

The present report describes application of advanced analytical methods to establish correlation between changes in human serum proteins of patients with coronary atherosclerosis (protein metabolism) before and after moderate beer consumption. Intrinsic fluorescence, circular dichroism (CD), differential scanning calorimetry and hydrophobicity (So) were used to study human serum proteins. Globulin and albumin from human serum (HSG and HSA, respectively) were denatured with 8 m urea as the maximal concentration. The results obtained provided evidence of differences in their secondary and tertiary structures. The thermal denaturation of HSA and HSG expressed in temperature of denaturation (Td, degrees C), enthalpy (DeltaH, kcal/mol) and entropy (DeltaS kcal/mol K) showed qualitative changes in these protein fractions, which were characterized and compared with fluorescence and CD. Number of hydrogen bonds (n) ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). Unfolding of HSA and HSG fractions is a result of promoted interactions between exposed functional groups, which involve conformational changes of alpha-helix, beta-sheet and aperiodic structure. Here evidence is provided that the loosening of the human serum protein structure takes place primarily in various concentrations of urea before and after beer consumption (BC). Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption. In summary, thermal denaturation parameters, fluorescence, So and the content of secondary structure have shown that HSG is more stable fraction than HSA.     

Non-random-coil behavior as a consequence of extensive PPII structure in the denatured state

Unfolded proteins may contain a native or nonnative residual structure, which has important implications for the thermodynamics and kinetics of folding, as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique—fluorescence correlation spectroscopy—to probe the denatured state of a set of repeat proteins containing an increasing number of identical domains, from 2 to 20. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random coil statistics. The origin of this unexpected behavior is traced to the presence of an extensive nonnative polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show that the experimentally observed effects of polyproline II on the size scaling of the denatured state can be well-described by simple polymer models. Our findings suggest a hitherto unforeseen potential of nonnative structure to induce significant compaction of denatured proteins, significantly affecting folding pathways and kinetics.     

NMR characterization of a peptide model provides evidence for significant structure in the unfolded state of the villin headpiece helical subdomain

The villin headpiece subdomain (HP36) is the smallest naturally occurring protein that folds cooperatively. The protein folds on a microsecond time scale. Its small size and very rapid folding have made it a popular target for biophysical studies of protein folding. Temperature-dependent one-dimensional (1D) NMR studies of the full-length protein together with CD and 1D NMR studies of the 21-residue peptide fragment (HP21) derived from HP36 have shown that there is significant structure in the unfolded state of HP36 and have demonstrated that HP21 is a good model of these interactions. Here, we characterized the model peptide HP21 in detail by two-dimensional NMR. Strongly upfield shifted C(alpha) protons, the magnitude of the 3J(NH,alpha) coupling constants, and the pattern of backbone-backbone and backbone-side chain NOEs indicate that the ensemble of structures populated by HP21 contains alpha-helical structure and native as well as non-native hydrophobic contacts. The hydrogen-bonded secondary structure inferred from the NOEs is, however, not sufficient to confer significant protection against amide H-D exchange. These studies indicate that there is significant secondary structure and hydrophobic clustering in the unfolded state of HP36. The implications for the folding of HP36 are discussed.     

Local structural preferences and dynamics restrictions in the urea-denatured state of SUMO-1: NMR characterization

We have investigated by multidimensional NMR the structural and dynamic characteristics of the urea-denatured state of activated SUMO-1, a 97-residue protein belonging to the growing family of ubiquitin-like proteins involved in post-translational modifications. Complete backbone amide and 15N resonance assignments were obtained in the denatured state by using HNN and HN(C)N experiments. These enabled other proton assignments from TOCSY-HSQC spectra. Secondary Halpha chemical shifts and 1H-1H NOE indicate that the protein chain in the denatured state has structural preferences in the broad beta-domain for many residues. Several of these are seen to populate the (phi,psi) space belonging to polyproline II structure. Although there is no evidence for any persistent structures, many contiguous stretches of three or more residues exhibit structural propensities suggesting possibilities of short-range transient structure formation. The hetero-nuclear 1H-15N NOEs are extremely weak for most residues, except for a few at the C-terminal, and the 15N relaxation rates show sequence-wise variation. Some of the regions of slow motions coincide with those of structural preferences and these are interspersed by highly flexible residues. The implications of these observations for the early folding events starting from the urea-denatured state of activated SUMO-1 have been discussed.     

Spectroscopic study of the structure of sucrose in the amorphous state and in aqueous solution

The structure of noncrystalline sucrose in the amorphous, solid state and in aqueous solution was investigated. Differences of structure of amorphous solid samples, the quenched-melt, and freeze-dried sucrose, are revealed by differential thermal analysis (d.t.a.) and from the Fourier-transform infrared (F.t.-i.r.) spectra. Factor analysis of the F.t.-i.r. spectra of aqueous solutions of sucrose shows the existence of at least two forms of the sucrose molecule. Analysis of ¹³C-n.m.r. spectra of amorphous and crystalline sucrose reveals a sensitivity of the fructosyl moiety to the morphology of the sample.     

Surface point mutations that significantly alter the structure and stability of a protein's denatured state.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.     

Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta-sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha-helix in the N-state. There is evidence for some fixed side-chain--side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.     

Solution structure of Eucommia antifungal peptide: a novel structural model distinct with a five-disulfide motif

The three-dimensional structure in aqueous solution of Eucommia antifungal peptide 2 (EAFP2) from Eucommia ulmoides Oliv was determined using (1)H NMR spectroscopy. EAFP2 is a newly discovered 41-residue peptide distinct with a five-disulfide cross-linked motif. This peptide exhibits chitin-binding activity and inhibitory effects on the growth of cell wall chitin-containing fungi and chitin-free fungi. The structure was calculated by using torsion angle dynamic simulated annealing with a total of 614 distance restraints and 16 dihedral restraints derived from NOESY and DQF-COSY spectra, respectively. The five disulfide bonds were assigned from preliminary structures using a statistical analysis of intercystinyl distances. The solution structure of EAFP2 is presented as an ensemble of 20 conformers with a backbone RMS deviation of 0.65 (+/-0.13) A for the well-defined Cys3-Cys39 segment. The tertiary structure of EAFP2 represents the first five-disulfide cross-linked structural model of the plant antifungal peptide. EAFP2 adopts a compact global fold composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Gly26-Cys30), and a three-strand antiparallel beta-sheet (Cys16-Ser18, Tyr22-Gly24, and Arg36-Cys37). The tertiary structure of EAFP2 shows a chitin-binding domain (residues 11-30) with a hydrophobic face and a characteristic sector formed by the N-terminal 10 residues and the C-terminal segment cross-linked through the unique disulfide bond Cys7-Cys37, which brings all four positively charged residues (Arg6, Arg9, Arg36, and Arg40) onto a cationic face. On the basis of such a structural feature, the possible structural basis for the functional properties of EAFP2 is discussed.