Short-range, Long-range and Transition State Interactions in the Denatured State of ACBP from Residual Dipolar Couplings

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1 doi: /j.jmb J. Mol. Biol. (2004) 339, Short-range, Long-range and Transition State Interactions in the Denatured State of ACBP from Residual Dipolar Couplings Wolfgang Fieber, Sigridur Kristjansdottir and Flemming M. Poulsen* Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, 1353 Copenhagen, Denmark *Corresponding author Residual dipolar couplings in the denatured state of bovine acylcoenzyme A binding protein (ACBP) oriented in strained polyacrylamide gels have been shown to be a sensitive, sequence-specific probe for residual secondary structure. Results supporting this were obtained by comparing residual dipolar couplings under different denaturing conditions. The data were analyzed using the program molecular fragment replacement (MFR), which demonstrated a-helix propensity in four isolated stretches along the protein backbone, and these coincide with the location of native helices. This is in full agreement with earlier findings based on secondary chemical shift values. Furthermore, N H residual dipolar couplings provided direct evidence for the existence of native-like hydrophobic interactions in the acid-denatured state of ACBP at ph 2.3. It was shown that replacement of the hydrophobic side-chain of residue Ile27 with alanine in helix A2 leads to large decreases of residual dipolar couplings in residues that form helix A4 in the native state. It is suggested that the Ile to Ala mutation changes the probability for the formation of long-range interactions, which are present in the acid-denatured state of the wild-type protein. These long-range interactions are similar to those proposed to form in the transition state of folding of ACBP. Therefore, the application of residual dipolar couplings in combination with a comparative mutation study has demonstrated the presence of precursors to the folding transition state under acid-unfolding conditions. q 2004 Elsevier Ltd. All rights reserved. Keywords: denatured state; dipolar coupling; native-like interaction; helix propensity; ACBP Introduction The introduction of residual dipolar couplings (RDC) from weak alignment in anisotropic media as a tool in nuclear magnetic resonance spectroscopy (NMR) opened a new gate towards the determination of three-dimensional structure of proteins. 1 Dipolar couplings depend on the angle defined by internuclear single-bond vectors and the external magnetic field. Therefore, valuable Supplementary data associated with this article can be found at doi: /j.jmb Abbreviations used: RDC, residual dipolar coupling; ACBP, acyl-coenzyme A binding protein; MFR, molecular fragment replacement. address of the corresponding author: fmp@apk.molbio.ku.dk information about the relative orientation of individual bond vectors can be obtained. The application of constraints derived from measurements of RDCs have improved the quality of NMR structures significantly, 2 and allowed for the determination of the relative orientation of protein domains, where NOE information was not sufficient. 3 Although the structural basis of RDCs in the disordered state is less well understood, recent studies have addressed this problem and examined these couplings in proteins under strongly denaturing conditions, 4 6 and in partially or unstructured peptides. 7,8 RDCs of the intrinsically denatured mutant of staphylococcal nuclease D131D have been shown to be well correlated even at high concentrations of urea, for multiple mutations of hydrophobic residues, and with /$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

2 1192 Residual Dipolar Couplings of Denatured ACBP truncations of N or C-terminal parts of the protein, respectively. 4,6 A similar long-range structure was supposed to be retained under each of these circumstances. It was proposed that these structures are formed by local steric and hydrogen bonding interactions between side-chains and the adjacent protein backbone rather than by longrange interactions. 6 The interpretation of these data, however, has been a matter of debate, as it was anticipated by the authors that the presence of dipolar couplings alone would prove the existence of long-range structure in the protein. On the contrary, in a recent study it has been shown that random chains show non-vanishing RDCs. 9 These results have set new standards in the interpretation of RDCs in denatured states, and it has been suggested to use them as a reference for the analysis of the contribution of residual structure to the observed dipolar couplings. In the present work, RDCs were used to investigate the structure in the denatured state of bovine acyl-coenzyme A binding protein (ACBP). ACBP is an 86 residue, single-domain protein that folds and unfolds according to an apparent two-state mechanism. 10 In its folded state, ACBP exhibits a characteristic four-helix bundle structure, stabilized by three identified hydrophobic minicores (Figure 1). 11 Mutation studies suggested that the formation of a native-like structure, including eight conserved hydrophobic residues in the N and C-terminal helices, is rate-limiting in the folding of ACBP. 12 To elucidate possible structural determinants for the folding mechanism in the denatured state, ACBP has been investigated at low ph and high concentrations of denaturant. Under both denaturing conditions, native-like secondary structure has been shown to form transiently. 13,14 Moreover, long-range interactions of ACBP in the presence of high concentrations of guanidinium hydrochloride were identified by site-directed spin labeling and paramagnetic relaxation enhancement. 13,15 Results Qualitative analysis of residual dipolar couplings of ACBP at ph 2.3 RDCs of ACBP under different denaturing conditions were obtained by partial alignment in stretched polyacrylamide gels. The observed N H dipolar couplings at ph 2.3 display a distribution between positive and negative values with maxima found at þ 11 Hz and 210 Hz, respectively. The relative sign of the coupling data gives rise to a pattern of distinct segments along the protein backbone (Figure 2(a)). Positive values are observed for four isolated stretches encompassing residues 5 10, 22 31, 53 60, and The strongest couplings occur in the C-terminal segment with values up to þ11 Hz, whereas the other three segments are shorter and display couplings not larger than þ7 Hz. Their positions coincide with the location of the four helices in the native state of ACBP. 11 At ph 2.3 the protein is entirely denatured with respect to the ph denaturing curve, but transient helix formation in these segments can still be observed by C a secondary Figure 1. Three-dimensional NMR structure of bovine ACBP. Helices are referred to as A1, A2, A3, and A4, starting at the N terminus. Residue Ile27, substituted by alanine in I27A, is shown in cyan. Figure 2. (a) N H dipolar couplings of ACBP at ph 2.3 as a function of residue position. Black bars denote the location of the four a-helices in the native structure of ACBP. (b) 13 C a secondary chemical shifts of ACBP at ph 2.3. Shifts were taken from Thomsen et al. 14 (c) Difference of N H dipolar couplings of ACBP at ph 2.8 and at ph 2.5 (ph 2.8 ph 2.5).

3 Residual Dipolar Couplings of Denatured ACBP 1193 chemical shifts 14 (Figure 2(b)). The effect is most pronounced in the C-terminal part, which forms helix A4 in the native state with shifts up to 2 ppm and an estimated helix population of approximately 30% of the time. Helices A1, A2, and A3 display lower secondary shifts, and the helix population was estimated to be 20% for helices A1 and A2, and approximately 10% for helix A3. Residual structure has also been observed by CD spectroscopy. 14 Analysis of the CD spectrum of ACBP at ph 2.3 yielded an overall a-helical content of 16% by comparing the values obtained to an a-helix reference base spectrum from eight proteins (data not shown). 16 This is in good agreement with the estimated overall a-helical content from NMR spectroscopy (15%). These results indicate a relationship between sign and size of the observed residual dipolar couplings and residual secondary structure. Dipolar couplings depend on the angular relationship between the individual bond vectors and the axes of a common reference frame referred to as the alignment tensor, 1 hence structural information from absolute values can generally not be achieved. Straight structural arrays such as a-helices, however, can be recognized by dipolar couplings of the same sign, as the amide N H bonds are aligned parallel with the helix axis and retain the same orientation with respect to the reference frame. A number of different procedures have been proposed to determine the structure of folded protein structures solely from RDCs 17 or by combining these with structure databases. 18,19 It is a prerequisite in these analyses to assume that chemical bonds have a defined and fixed orientation relative to a single reference frame, and the alignment of the protein can be described by a Saupe order matrix. 20,21 For a protein under denaturing conditions this is not applicable. In the completely unfolded state, the bond vectors each sample their own conformational space and there is no common alignment tensor. Instead, in a highly simplified analysis, the dipolar couplings of ACBP at ph 2.3 have been analyzed using molecular fragment replacement (MFR). 18 In applying this method, the molecule is treated as a single conformational state, and the RDCs in the denatured state of ACBP are considered to correspond to a single set of vector orientations. This may not represent torsion angles that match physically or biologically realistic conformations, but it could help to identify protein segments that adopt a relatively stable structure in the time average. The MFR computer program fits experimental dipolar couplings and chemical shift values of seven residue fragments to fragments from the RCSB Protein Data Bank. The corresponding f,c backbone torsion angles from the best fitting fragments can then be used to build an initial structure of the molecule under investigation. For ACBP in the denatured state at ph 2.3, the 83 N H signals in the heteronuclear single-quantum coherence (HSQC) spectrum have been assigned, 14 and 81 N H, 70 C a C 0,70C 0 N, and 75 C a H a RDCs were determined in stretched polyacrylamide gels at ph 2.3 and used in the fitting process. Attempts to measure RDCs in other media such as polyethylene glycol C8E5 failed because acid-denatured ACBP interacted with these media. An analysis of the f and c angles in the best fitting fragments obtained in the MFR calculations showed that nearly all residues that have negative N H dipolar couplings fall in a single cluster in the b-sheet region. At positions with positive N H dipolar couplings, a second cluster of f and c angles in the a-helix region can be observed, and for several of these positions even a single cluster in this region. However, due to the degeneracy of RDCs, the use of data from only one alignment medium can result in false positive hits, 18,19 and this impedes the usage of these fitted fragments for the building of the whole protein backbone of ACBP at ph 2.3. The best fitting fragments were therefore analyzed individually by their RMSD to a modeled a-helix (Figure 3). Throughout the protein sequence, the MFR analysis of the entire set of backbone RDCs of acid-denatured ACBP has identified helical-like peptide fragments as the best fit, particularly in the regions that form a-helices under native conditions. This is seen most prominently in the central region of the peptide segment that forms helix A4 in the native state, with RMSD values between 0.2 Å and 0.7 Å. Thus, the MFR analysis shows unambiguously the presence of a-helix structure in the helix A4 segment in the denatured state of ACBP at ph 2.3. The fragments fitted to the central residues of helices A1, A2, and A3 also show better than average RMSD values to the model a-helix. However, the corresponding structures do not have complete helical character. This is in agreement with earlier findings that, in contrast to helix A4, helices A1, A2, and A3 are shorter and exhibit lower helix-forming propensity in the denatured state of ACBP. 13,14 Figure 3. RMSD values between the best-fitting seven residue fragments from MFR analysis of residual dipolar coupling of ACBP at ph 2.3 and a modeled a-helix. Each fragment is displayed at its middle position.

4 1194 Residual Dipolar Couplings of Denatured ACBP The RDC data, therefore, seem to reflect that restricted conformational sampling due to fluctuating secondary structure brings the respective N H vectors into parallel alignment along the transiently formed helix axis in a certain proportion of time. This is apparently what is reflected in the observable pattern of dipolar couplings even in the ensemble average. Residual dipolar couplings of ACBP under different denaturing conditions The relationship between RDCs and the degree of formation of transient helical structure was examined for denatured states at ph 2.5 and ph 2.8. The denatured state is populated 99.6% at ph 2.5 and 95% at ph 2.8, respectively, but slow chemical exchange between folded and unfolded states results in two observable signals for each residue in the respective HSQC spectrum. This allows for the selective analysis of signals only from the unfolded states at these conditions. It is known from ph titration experiments that ACBP folds in a cooperative transition between ph 2.4 and ph 3.6, 14 where the amount of secondary and tertiary structure of the denatured state increases with increasing ph. The N H RDCs measured at both ph values show the same pattern as that observed at ph 2.3, but the magnitude of the couplings in the four a-helical regions increases with increasing ph (Figure 2(c)), notably in the helix A4 segment (up to 5 Hz) and to a smaller extent in the other three helix segments (up to 2.5 Hz). Couplings in the loop regions, on the other hand, remain mainly unaffected. In the presence of 2.5 M GuHCl at ph 5.3 (denatured state populated 98%) only negative couplings were detected (Figure 4(a)). All C a secondary chemical shifts under these conditions are found to be shifted,0.4 ppm to lower field compared to random coil values. 22 Only residues in helices A2 and A4 show slightly higher values, indicating the presence of transient helical structure in these two segments (Figure 4(b)). This result is similar to what was observed for the I86C mutant of ACBP at 1.9 M GuHCl. 13 A further loss of helical structure at higher concentrations of denaturant in this mutant protein was evident from 15 N chemical shifts, where the changes appeared to be largest in helices A2 and A4. By increasing the concentration to 4 M GuHCl, where the denatured state is,100% populated, RDCs of wild-type ACBP became even more negative, with the largest effect in the two segments corresponding to helices A2 and A4 in the native state (Figure 4(c)). The two sets of comparisons suggest a correlation between the changes in RDCs of the helix segments of denatured ACBP with the change in denaturing conditions both with ph and GuHCl. In this respect, it is important to emphasize that the technical reliability of these comparisons were facilitated by a very high reproducibility of the Figure 4. (a) N H dipolar couplings of ACBP at ph 5.3 and in the presence of 2.5 M GuHCl. Black bars denote the location of the four a-helices in the native structure of ACBP. (b) 13 C a secondary chemical shifts of ACBP at ph 5.3 and in the presence of 2.5 M GuHCl. (c) Difference of N H dipolar couplings between 2.5 M GuHCl and 4 M GuHCl (2.5 M 4 M). alignment in polyacrylamide gels as monitored by the quadrupolar splitting of the 2 H resonance of the solvent of 15.3(^0.3) Hz for all the samples of different preparations and denaturing conditions. The origin of the specific changes in the residual dipolar couplings may be due to global changes in the denatured state, which affect the overall alignment. Alternatively, they may be caused by local changes with an origin in the change in an equilibrium constant, which increases the concentration of one particular conformer. Comparison of N H dipolar couplings of ACBP measured at ph 2.5 and ph 2.8 shows that the two datasets correlate very well ðr ¼ 0:984Þ (Figure 5(a)). Likewise, both datasets at ph 2.5 and ph 2.8 correlate very well to that obtained at ph 2.3 (R ¼ 0:987 and R ¼ 0:986, respectively, data not shown). This suggests that the observed coupling differences are an effect of local origin rather than a global structural effect. A comparison of the RDCs at 2.5 M and 4 M GuHCl shows a weaker but still reasonable correlation ðr ¼ 0:876Þ. A similar comparison involving only the segments of the peptide chain that are in non-helical segments in the native state, showed that their RDCs were practically all independent of changes in the denaturing conditions with respect to both ph and GuHCl. (Figure 5(a) and (b)). A clear difference between the acid-denatured states at ph 2.3 and at 2.5 M

5 Residual Dipolar Couplings of Denatured ACBP 1195 denatured ACBP irrespective of the different denaturing conditions, and that the helix random coil equilibrium in the presence of GuHCl is shifted more towards the random coil conformer than in the acid-denatured form. Structural changes in the I27A mutant observed by RDCs Figure 5. Scatter plots of N H dipolar couplings of ACBP at various denaturing conditions. Black and red circles depict residues located in the four a-helices and in the loop regions, respectively, in native ACBP. Linear regression for all residues (black) and non-helical residues (red) is depicted. (a) ph 2.5 versus ph 2.8. (b) 2.5 M GuHCl versus 4 M GuHCl. (c) 2.5 M GuHCl versus ph 2.3. The presence of potential long-range interactions in denatured ACBP was investigated by sitedirected mutagenesis. Residue Ile27 is part of helix A2 and engaged in one of the three hydrophobic mini-cores identified in folded ACBP, 11 where it has stabilizing van der Waal s interactions to side-chains of residues Val12 in helix A1 and to Val77 in helix A4 (Figure 1). These two residues are engaged in the transition state of the folding of ACBP, 12 and it has been shown that Ile27 is a transition state residue, as revealed by a w-value analysis, showing a w-value of 0.3 (data not shown). Like the two other transition state residues, Ile27 is a conserved hydrophobic residue in most of the sequences of ACBP from different organisms. 23 The mutant I27A was used to probe for the structure formation in the denatured state by RDCs. N H dipolar couplings measured for I27A show the same pattern as that observed for the wild-type. However, a remarkable reduction in the couplings is observed (Figure 6(a)) in the vicinity of the mutated residue in helix A2 and, to an even greater extent, in the distant helix A4. GuHCl and their behavior in the alignment medium is reflected by the overall weak correlation of residual dipolar couplings ðr ¼ 0:673Þ measured at the two conditions. A comparison of the RDCs for the non-helical residues shows a better correlation ðr ¼ 0:854Þ, which may reflect a trend, that these segments are equally unstructured at both conditions (Figure 5(c)). Finally, it is noted that for both the acid and denaturant-unfolded states, the change of conditions that favor the formation of transient helical structure also change the size of RDC in those segments, which have helical structure in the native state. The change is seen to be either towards larger positive values in the acid-denaturing study or towards smaller negative values in the GuHCl study. This is in agreement with the MFR analysis, which showed that helical residues contribute to positive dipolar couplings in the denatured state. It suggests also that the helix conformers in the helix regions remain the most abundant in Figure 6. (a) Difference of N H dipolar couplings between wild-type ACBP and I27A at ph 2.3 (ACBP I27A). Black bars denote the location of the four a-helices in the native structure of ACBP. (b) Scatter plot of N H dipolar couplings of I27A versus wild-type ACBP. Black and red circles depict residues located in the four a-helices and in the loop regions, respectively, in native wild-type ACBP. Linear regression for all residues (black) and non-helical residues (red) is depicted.

6 1196 Residual Dipolar Couplings of Denatured ACBP Residues in helix A1 are affected weakly, whereas the remaining segments show the same dipolar coupling values as in the wild-type protein. The N H dipolar couplings between wild-type ACBP and I27A at ph 2.3 are highly correlated ðr ¼ 0:979Þ (Figure 6(b)). Moreover, non-helical residues display nearly identical values ðr ¼ 0:985Þ. This indicates again that the major structural changes occur in the segments of the peptide chain forming a-helices under native conditions. It was shown above that the reduction in N H dipolar couplings may be associated with a reduction in segmental helix-forming propensity. In the denatured state of the I27A mutant, reductions of dipolar couplings were observed for residues in helices A1, A2 and A4 relative to the denatured wild-type protein. This corresponds to a decrease in transient helical formation in these segments of the mutant protein. The CD spectrum of the mutant protein corroborates these findings. It differs from that of the wild-type protein by a smaller magnitude of the absorption band around 222 nm, and the calculated overall helical content is 14% (data not shown). 16 These differences are due to the isoleucine to alanine mutation, and they indicate the existence of interactions between Ile27 in the wild-type protein and residues in helices A1 and A4. These are exactly the nativelike interactions that were suggested from protein engineering studies and w-value analysis to be part of the folding transition state, including the side-chains of Ile27 in A2, Val12 in A1 and Val77 in A4. The present results are in good agreement with this and suggest the existence of transient precursors to the folding transition state and the associated helix formation in the three helix segments under denaturing acidic conditions. Discussion Here, RDCs have been used to study the denatured state of ACBP. The N H RDCs of ACBP are negative in sign all along the protein backbone when denatured in 2.5 M GuHCl, a condition where only very little secondary structure is present. By further increasing the concentration of denaturant, they generally shift to larger negative values. Recently, Annila and co-workers proved the existence of non-vanishing RDCs even in random chains, and established the basis for a better understanding of the origins of RDCs in denatured states of proteins. 9 Both calculated and simulated RDCs are negative and display a bellshaped distribution along the protein backbone with the highest values at the center position. The size of the couplings is inversely proportional to the chain length, and dipolar couplings are predicted to level off to zero at infinitely long chain lengths, where the distribution of the structural ensemble becomes more spherical. In the light of this analysis, the non-uniform distribution of N H dipolar couplings along the protein backbone of ACBP at 2.5 M GuHCl suggests that under these conditions ACBP is not a random coil but presumably reflects the presence of residual structure, which has been seen in other studies. 13,15 The observation that the negative RDCs increased when accessing a more denatured state of ACBP with less residual structure is an unexpected result, since, according to the model proposed by Annila and co-workers, this is not predicted. It suggests that the origin of the alignment of the unfolded state under these denaturing conditions is more complex than just the anticipation of a completely random structure. In a study of ACBP at high concentrations of GuHCl (1.6 M, 1.9 M, 3 M), spin-label relaxation enhancement data and restrained computer simulations using a system of non-interacting replica of the protein were used to obtain ensembles of conformations that represent the denatured state. 15 The calculated radii of gyration of the ensemble increase with the concentration of denaturant, reflected also by a shift of the size distribution pattern towards more extended structures and an increasing proportion of highly unfolded molecules (K. Lindorff-Larsen, personal communication). As the molecular alignment tensors of these highly asymmetric and extended molecules are very large, the respective dipolar coupling values can be very large, too, and would contribute predominantly to the average residual dipolar couplings of the ensemble. This could be one possible explanation for the observed behavior of dipolar couplings as a function of denaturant concentration. However, it may require additional experiments and simulations to elucidate the molecular determinants of conformational averaging and alignment in denatured proteins. In contrast to the measurements at high concentrations of GuHCl, the N H dipolar couplings of ACBP denatured at ph 2.3 show a distinctive, sequence-specific pattern. Positive values for N H dipolar couplings were observed in segments of native a-helices that contain a significant amount of residual helical structure, 14 and negative values were observed in the loop regions between the regions forming helices in the native state. By measuring four different dipolar coupling data sets and fitting the results to fragments from the Protein Data Bank by MFR software, 18 it could be demonstrated unambiguously that the positive couplings arise from a-helical conformations. The analysis suggests, therefore, that they form in such a proportion of time that they can be observed as an array of consecutive positive couplings even in the time-average of the ensemble. Similar to previous observations, helix A4 appears to be the longest and most stable a-helical stretch in the denatured state of ACBP. 13,14 The positive RDCs in the helix segments seem to scale with the amount of residual structure, as their magnitude is reduced when the ph of the protein solution was lowered from 2.8 to 2.5. It is known from earlier studies on denatured ACBP that the helical content scales

7 Residual Dipolar Couplings of Denatured ACBP 1197 with the ph of the solution, and a cooperative transition between folded and unfolded states has been observed between ph 2.4 and ph Residues between the helix segments in the peptide chain, on the other hand, are invariant or display lower than average coupling differences between ph 2.5 and ph 2.8. Taking these data into consideration, they are in excellent agreement with the results from RDC studies on the free S-peptide of ribonuclease A. 8 Residues in the segment of the native a-helix are opposite in sign to residues at the N and C termini of the peptide. They increase in magnitude when the helix is stabilized by addition of salt and by low temperature, respectively, whereas the other residues roughly retain their dipolar coupling values. It has been demonstrated convincingly that the observed N H dipolar couplings are compatible with transient a-helical structure in the peptide. This is similar to what was shown here for denatured ACBP at ph 2.3, however, the reported values of the respective RDCs have opposite sign. The derivation of N H residual dipolar couplings in the present work takes into account the negative gyromagnetic ratio of 15 N. One-bond scalar couplings measured in an isotropic sample and the total couplings obtained from an aligned sample were determined by their absolute (positive) values in the spectra. RDCs were calculated as the difference in couplings between isotropic and aligned samples (isotropic aligned), 24 whereas they were calculated the other way round for the S-peptide (aligned isotropic). 8 On the other hand, N H dipolar couplings for S-peptide and ACBP have been obtained from different alignment media, a mixture of polyoxyethylene 5 octyl ether (C8E5) and 1-octanol, and polyacrylamide gels, respectively. Yet, the obstructing plane of the bicelles as well as the long axis of the elliptical cavity in the gel are parallel with the external magnetic field, 25,26 and experimental results demonstrated that in the absence of interactions between the solute and the media the sign of dipolar couplings in both media is retained (W.F., unpublished results). 5 If this relation is valid for the S-peptide, RDCs can be compared directly to coupling data presented here for denatured ACBP by scaling the reported values from the S-peptide by a factor of 21 to compensate for the different determination of RDCs. This would result in positive N H dipolar couplings of helical residues, analogous to what was observed in acid-denatured ACBP. The existence of residual structure is evident also when comparing the data to the calculated dipolar couplings for a random flight chain. 9 The distribution of N H dipolar couplings of ACBP at ph 2.3 is no longer uniformly negative in sign but rather grouped sequence-specifically into segments of positive and negative signs. However, the molecular determinants of alignment and conformational averaging that lead to the observed sign dependence are not known. Substitution of the hydrophobic side-chain of Ile27, a residue proposed to be involved in the transition state of the folding reaction, resulted in a decrease of residual structure in the direct vicinity of the residue as well as, to an even greater extent, in remote helix A4. In the native state of ACBP, Ile27 participates extensively in hydrophobic stabilization of the structure by forming interactions to residues in helix A1 and A4. Therefore, the observed effect indicates native-like interaction in the denatured state at ph 2.3. It could be demonstrated in a study of the long-range interactions in the GuHCl-denatured state of ACBP that the strongest interactions are formed between individual residues in the helix segments of A2 and A4. 15 This important interaction has now been demonstrated by spin-label relaxation studies, transition state analysis, and here by comparing RDC measurements in mutant and wild-type ACBP. Other studies have shown that the contribution of one residue to stabilizing hydrophobic interactions in the denatured state can be very large. By mutating a single tryptophan residue (W62G) in denatured lysozyme at ph 2, all clusters of residual structure observed in the wild-type protein were destabilized substantially. 27 Conclusion It has been demonstrated that RDCs can provide important information about residual structure in different states of denaturation of ACBP. Specifically, N H dipolar couplings are a very efficient tool for the study of transient helix formation. This has been shown by measurements of the increment in dipolar coupling from spectra recorded at different denaturing conditions or by comparing dipolar couplings in the spectra of mutant proteins. In the case of ACBP, sequencespecific structural changes could be revealed with great sensitivity. The use of RDCs in combination with sitedirected mutagenesis has been shown to be an important tool for the identification of transient long-range interactions. Here, residual dipolar couplings supported the proposed transition state interactions in the folding process of ACBP. Materials and Methods 15 N and 13 C, 15 N-labeled bovine wild-type ACBP and 15 N-labeled I27A mutant were expressed and purified as described. 28 RDCs were achieved by strain-induced alignment of 7% (w/v) polyacrylamide gels (acrylamide/bisacrylamide, 30 : 1, w/w). 25,29,30 After polymerization in a cylindrical gel chamber (ID 6.0 mm) overnight the gels were washed with water or the appropriate buffer solution for one to two hours. The protein solution was prepared by dissolving freeze-dried 15 Nor 13 C, 15 N-labeled bovine ACBP in water (10% 2 H 2 O). In the case of GuHCl-denatured ACBP, the solution was buffered by 20 mm sodium acetate, and the appropriate

8 1198 Residual Dipolar Couplings of Denatured ACBP amount of GuHCl was added from a stock solution. The gel was placed into a 2 ml Eppendorf tube and covered with approximately 300 ml of the protein solution and incubated for four to five hours to ensure reasonable diffusion of the protein into the gel. More time was needed in the presence of high concentrations of denaturant. Protein concentrations in the gels varied between 0.3 mm and 0.7 mm. A special apparatus (NewEra Enterprises, Inc.) for the preparation of stretched polyacrylamide gels was used. 30 NMR experiments were recorded at 298 K on Varian Unity Inova 750 MHz and 800 MHz instruments. The magnitude of the solvent 2 H quadrupolar splitting was used to monitor the alignment. The N H dipolar couplings were determined from spin-state-selective (S 3 CT) filtered 15 N-HSQC experiments 31,32 or from standard three-dimensional HNCO experiments, where 15 N decoupling during acquisition was omitted. N H residual dipolar couplings were calculated as the difference between scalar couplings measured in an isotropic sample and total couplings measured in an oriented sample. C a C 0,C 0 N 33, and C a H a34 couplings were determined from three-dimensional HNCO-based experiments. All spectra were processed and analyzed using nmrpipe 35 and NMRView 36 software. An in-house program was used to determine the scalar and dipolar couplings. The standard deviation was 0.38 Hz for N H, 0.41 Hz for C a C 0, 0.17 Hz for C 0 N, and 0.81 Hz for H a C a dipolar couplings. Assignment of ACBP at 2.5 M GuHCl and 4 M GuHCl was based on the chemical shift values of the I86C mutant at 1.9 M GuHCl, 13 whereas assignment at ph 2.5 and ph 2.8 was based on chemical shift values of ACBP at ph Secondary chemical shifts are the differences between observed values and random coil values from the literature, 22 where aspartate and glutamate residues were not taken into account for measurements at ph 2.3. Helix populations were calculated by normalizing C a secondary chemical shifts at ph 2.3 to the respective values in the folded state of ACBP at ph and averaging over all residues located in the respective helical segment. The program MFR was used in its default form. 18 N H, C a C 0, C 0 N, and C a H a dipolar couplings obtained from alignment in stretched polyacrylamide gels were used in the analysis. A model structure for a short a-helix was generated with the program InsightII (Molecular Simulations, Inc.) and energy minimized using built-in Discover software. The CD spectra of ACBP at ph 2.3 were recorded on a Jasco J-810 CD spectrometer at 298 K. Quartz cells with 0.1 cm path-length were used. Analysis of the CD spectrum was performed by comparing the measured spectrum to an a-helix reference base spectrum from eight proteins. 16 Acknowledgements W.F. is the recipient of an Erwin Schrödinger Fellowship (J2229-B07) from the Austrian Science Foundation (FWF). 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