Chapter Study on Highly-Populated Helical Conformations in the Partially-Folded hNck2 SH3 Domain Part of this chapter has been published in: Liu J, Song J. NMR Evidence for Forming Highly-Populated Helical Conformations in the PartiallyFolded hNck2 SH3 Domain. Biophys J. 2008 Jul 3. PMID: 18599634 88 4.1 Introduction Many proteins fold into unique three-dimensional scaffolds composed of spatially organized polypeptide fragments that possess different secondary structures, including the α-helix, β-sheet, reverse turns, and loops. Some proteins were found to fold into their native states via intermediates, and in particular, the partially folded states termed “molten globule” were studied extensively (Kim, P. S., 1982; Kim, P. S., 1990; Kuwajima, K., 1989; Ptitsyn, O. B., 1995; Jennings, P. A., 1993; Colón W, Roder H., 1996; Song, J., 1998; Song, J., 1999; Wei, Z., 2005; Daggett, V., 2003). Many proteinfolding models emphasize the hierarchical formation of the native-like substructures during the folding process. On the other hand, recent studies of several proteins, and particularly β-lactoglobulin, implied that the folding of β-proteins may follow a nonhierarchical mechanism in which two major transitions are essential to reach the final native β-structure. More specifically, the first transition is involved in the collapse of the random-coil-like polypeptide chain into a non-native helical intermediate state mainly specified by local interactions, whereas the second transition is associated with transformation into the native β-structure, with the helical conformation disrupted by long-range interactions (Kuwajima, K., 1996; Hamada, D., 1996; Hamada, D., 1997; Arai, M., 1998; Fujiwara, K., 1999; Chikenji, G., 2000; Kuwata K, 2001). However, the conformational and dynamic properties of such non-native helical states still remain poorly understood. Moreover, the sequence determinants for transformation from the helical intermediate into the native β-structure have not been identified. So far, more than 4,000 SH3 modular domains have been identified in a variety of organisms. The SH3 domains, consisting of about 60 residues without disulfide bridge, play a critical role in 89 transmitting as well as integrating cellular signals, mainly by binding to proline-rich short motifs (Mayer, B. J., 2001; Musacchio, A., 2002; Liu J, 2006). From structural point of view, all SH3 domains share a common β-barrel fold comprising five β-stands, which are organized into two β-sheets. The first, the last and the first half of the second β-strand constitute the first β-sheet, whereas the second half of the second β-strand, together with the third and fourth β-strands constitutes the second β-sheet. The second β-strand seems to play a critical role in coordinating the two β-sheets together into a classical SH3 fold (Fig. 4.1A). The 380-residue adaptor protein Nck2, which is composed of three SH3 domains and one C-terminal SH2 domain, in vivo, functions to coordinate the signalling networks critical for the organization of the actin cytoskeleton, cell movement, or axon guidance, by connecting membrane receptors to the multiple intracellular signalling networks in a “Tyr(P)/SH2/SH3/effector” manner (Li, W., 2001; Ran, X., 2005). The NMR structure of the first Nck2 SH3 domain was previously determined (Park S, 2006), and the structures of the second and third SH3 domains as well as the SH2 domain were determined by our lab (Ran, X., 2005; Liu J, 2006). SH3 domain is an ideal model system for investigating the folding mechanism of β-proteins due to its small all-β fold and the absence of disulfide bridge. Unexpectedly, we discovered, without any co-solvent or stabilizer, that the wild type form of the first Nck2 SH3 domain could be reversibly converted into a stable helical state of equilibrium at pH 2.0, as detected by circular dichroism CD spectroscopy. To gain further insights, we performed extensive CD and NMR investigations, and the results not only presented us an NMR conformational and dynamic view of this non-native helical state, but also led to the further identification of a four-residue sequence that appears to play an important role in transforming the helical 90 state into the native SH3 fold. Recently, a study was published demonstrating that a nonnative helical intermediate was also populated in the kinetic refolding of the Src SH3 domain (Li, J.S., 2007). Another article from the same group showed that the Ala45-Gly mutant of the Src SH3 domain could also form a stable helical intermediate at equilibrium, which were studied by CD, fluorescence, and x-ray scattering (Li, J., 2007) 4.2 Materials and methods 4.2.1 Expression and purification The mutant of L25A-W26A-L27A-L28A was obtained by the same method as wild type, with the middle two primers changed to the corresponding primers encoding the four Alanine residues. The expression, 15 N/(15N/13C) labelling and purification of the wild type were performed as described in Common Materials and Methods Chapter. The refolding of the wild type SH3 was carried out by slowly titrating with a 5mM phosphate buffer until the pH value 6.5. An NMR sample was then concentrated to ~1mM for further NMR experiments. The mutant was purified as described above and performed dialysis in a 1mM phosphate buffer. The sample was then concentrated to ~1mM. 4.2.2 CD and NMR experiments Concentrations of the proteins were around 15 μM in a mM phosphate buffer. The farUV spectra of the mutant SH3-1 were acquired in the range of 190-260nm at different pH values, at intervals of pH 0.5. The wild type SH3 far-UV spectra were acquired at pH 2.0, pH 4.5 and 6.5. 91 NMR experiments included 1H NOESY, TOCSY (250ms and 350ms), 1H-15N HSQC, H-15N HSQC-TOCSY, 1H-15N HSQC-NOESY, HNCACB, CBCA(CO)NH, HCCH- TOCSY and HCCH-NOESY for the SH3-1 wild type at pH 2.0. All these experiments except HCCH-TOCSY, HCCH-NOESY were also carried out for the mutant. T1 and T2 experiments and a {1H}-15N heteronulear NOE experiment were also carried out for the wild type at pH 2.0 and the mutant at pH 6.5. To test whether SH3-1 WT at pH 2.0 is recoverable to a native state, the lyophilised powder was first dissolved in pure water (around 10ml) with a pH value ranging from 2.0 to 2.5. Subsequently, the solution was gradually titrated by a mM phosphate buffer. The titration was stopped when the pH value reached 6.5. The sample was finally concentrated to ~1 mM. The prepared sample was further used to HNCACB, CBCA(CO)NH and relaxation experiments. 4.2.3 Structure modelling of wild type (pH 2.0) and 4AlaMut Dihedral angles used for structure modelling were predicted by the TALOS program (Cornilescu, G., 1999), based on the chemical shifts of HA, CA and CB for the wild type at pH2.0 and HA for the mutant at pH 6.5. 4.2.4 Relaxation experiments 15 N T1 values for the wild type at pH 6.5 were obtained by fitting HSQC spectra recorded with different relaxation delays of 10, 400, 100, 300, 200, 350 and 250 ms. 15 N T2 values were determined with relaxation delays of 10, 30, 45, 60, 75, 90 and 150ms for the wild type at pH 6.5; For the wild type at pH 2.0, relaxation delays for 15 N T1 are: 10, 700, 100, 92 600, 200, 500, 300, 400 ms; for 15 N T2: 10, 60, 100, 130, 160, 200, 230, 260 ms. For 4AlaMut, delays for 15N T1 are: 10, 350, 50, 300, 100, 400, 200 ms while for 15N T2: 10, 30, 60, 90, 120, 140, 160. {1H}-15N steady-state NOEs were obtained by acquiring spectra with and without 1H presaturation with a duration of s plus a relaxation delay of s at 800 MHz. 4.3 Result 4.3.1 Bioinformatics and CD characterization Previously, a high-resolution NMR structure was reported for the first human Nck2 SH3 domain (Park S, 2006), which unambiguously showed that it adopts the classical all-β fold common to all SH3 domains. On the other hand, as shown in Figure 4.1A and B, prediction of its secondary structures by several computational programs, including GOR4 (Garnier, J., 1996), PHD (Rost, B., 1994), Predator (Frishman, D., 1996), SHIMPA96 (Levin, J., 1997) and SOPMA (C Geourjon, 1995), suggested that a large H H portion of the SH3 domain had a high intrinsic propensity to form helical conformations. In particular, the results of GOR4 and Predator indicated even higher percentage of helical conformation than the β-sheet in the SH3-1 domain (Fig.4.1B). However, as empirical information including the tertiary structure from the Protein Data Bank was utilised in these programs to improve the prediction accuracy, we used Helix2, which implements the Lifson-Roig helix-coil transition theory and only uses the interactions between adjacent residues (Rohl, C. A., 1996; Rohl, C. A., 1998). The prediction of Helix2 also showed that the SH3-1 domain contained a 21% helix fraction. However, the prediction by AGADIR (Munoz, V.1, 1995; Munoz, V.2, 1995) only yielded 2.2% and 93 1.1% helix fractions at pH 6.5 and 2.0, respectively. In general, it is widely accepted that helix formation is mainly driven by local interactions (Rohl, C. A., 1996; Munoz, V., 1995), whereas β-sheet formation largely depends on long-range interactions and thus is context-dependent (Minor D. L Jr, 19941; Minor D. L Jr2). The discrepancy between the NMR structure and secondary structure prediction results inspired us to speculate that at least for the first hNck2 SH3 domain, the formation of the all-β SH3 native fold might be extensively driven by specific long-range interactions, which also function to override the intrinsic helix-formation propensity. Therefore, destabilization of the tertiary packing would trigger a conversion of the all-β SH3 fold into a helical state. As shown in Fig4.2, at pH 6.5, the far-UV CD spectrum of wild type SH3 domain demonstrates some unique characteristics (Venyaminov, S. Y., 1996). The minimal ellipticity at 203 nm and maximal ellipticity at 227 nm suggest the presence of some polyproline II structure in the native state of the protein. Although all SH3 domains adopt the same three-dimensional fold, they have diverse far-UV CD spectra and dynamic properties, as previously documented (Wales, T. E., 2006; Liu J, 2006). The origin of these unique properties is not well-understood, and some might result from the existence of the very long (>10 residues) and unique loop in all SH3 domains, which doesn’t adopt regular secondary structure, but makes extensive tertiary contacts with other parts of the SH3 domains (Liu J, 2006). By contrast, it appeared that at pH 2.0, the SH3 domain switched into a partially folded state containing some residual helical conformation. Variable secondary-structure fractions are given in the analysis of CD spectra by different programs: 15% helix, 35% turn/strand, and 5% random coil by CONTINLL; 12% helix, 46% turn/strand, and 42% random coil by SELCON3; and 5% helix, 59% turn/strand, and 36% random coil by 94 Figure 4.1 Bioinformatics analysis of secondary structures. A) Secondary structure prediction of the first hNck2 SH3 domain by computational programs GOR4, PHD, Predator, SIMPA96 and SOPMA. “E” stands for the β-strand, “H” stands for helix and “C” stands for the random coil. The four residues “LWLL” identified here are highlighted in yellow. The SH3 family alignment is shown at the bottom. “l” stands for aliphatic; “p” stands for polar. B) Bar plots for the secondary structure contents predicted by five different programmes. 95 Figure4.2 Far-UV CD characterization of the first hNck2 SH3 domain Far-UV CD spectra for the wild type at pH 6.5 (black); wild type at pH 2.0 (gray); 4AlaMut at pH 6.5 (dotted line) and 4AlaMut at pH 2.0 (broken line). Inset: pH-induced conformational changes of the wild type as monitored by ellipticity at 222 nm. 96 CDSSTR (Sreerama, N., 2000). Furthermore, the fraction of helix was estimated at ~11% by assuming the coexistence of the helix and random coil (Li, J.S., 2007). Detailed CD investigations also revealed that the pH-induced conformational change was reversible, with the transition occurring from pH to 2, as monitored at 222 nm. Further subsequent NMR studies were conducted extensively and these results confirmed (consistent with the CD observations) that the first Nck2 SH3 domain switched into a highly populated helical state at pH 2.0. This result raised the question of whether a sequence region could be identified in the first hNck2 SH3 domain that served as a switcher to transform the helical state into the native SH3 fold. In other words, mutation of this region could be anticipated to keep the SH3 domain permanently in the helical state. To address this question, multi-sequence alignment of large number of SH3 sequences selected from all subfamilies was performed, leading to the identification of a four-residue region, Leu25Trp26-Leu27-Leu28, on the second β-strand (Figure4.1A). Three of these residues were highly conserved in the majority of sequences. Further prediction of secondary structures of a variety of SH3 sequences by different programs also showed that this four-residue region always formed a β-strand. Therefore, we experimentally constructed a mutant with these four residues changed to Ala residues. Surprisingly, as seen in Figure4.2, the 4AlaMut protein showed very similar CD spectra at pH values of 6.5 and 2.0, suggesting that it completely lost the ability to undergo the pH-induced conformational changes observed in the wild type SH3 domain. Importantly, even at pH 6.5, 4AlaMut had a farUV CD spectrum similar to that of the wild type at pH 2.0, indicating that 4AlaMut were in a partially folded state bearing a similar fraction of the helical conformation even at a neutral pH. Far-UV CD spectra were also acquired at different protein concentrations in 97 Figure 4.3 1H-15N NMR HSQC spectra for three forms of SH3-1: A) Wild type at pH 2.0; B) 4AlaMut at pH 6.5; C) Wild type at pH 6.5. All spectra were acquired on an 800 MHz NMR spectrometer at 278 K. The sequential assignments were labelled for all spectra. 99 studied here adopts the same solution structure as previously determined (Park S, 2006). Because NMR chemical-shift deviations from those expected for random coils are very sensitive indicators of protein secondary structure (Merutka G, 1995; Schwarzinger S, 2000; Wishart D. S., 1998; Avbelj F., 2004), Cα and Hα chemical-shift deviations from the random-coil values previously reported were calculated (Merutka G, 1995; Schwarzinger S, 2000). As clearly indicated by the Cα deviation (Figure4.4A), the wild type SH3 domain adopts a β-like secondary structure at pH 6.5, whereas upon lowering the pH value to 2.0, it switched into a helical conformation. More specifically, it appeared that this β-to-helix transition occurred in the region of the majority of the RT-loop, the second and third β-strands. As shown in Figure4.3B, the large Hα deviations confirmed the formation of highly populated helical fragments with a similar pattern in both the wild type at pH 2.0 and 4AlaMut at pH 6.5. The NMR conformational shift was extensively used to estimate the population of the secondary structure in partially folded peptides and proteins (Ramirez-Alvarado, 1996; Jourdan M, 2000). Recently, it was shown that conformational shifts of amino acids were highly dependent on the extent of solvent exposure (Avbelj F., 2004). Statistically, the chemical-shift deviations of residues exposed to solvent are smaller than those of buried residues in the same secondary structure. According to their narrow HSQC dispersions, the wild type SH3-1 domain at pH 2.0 and 4AlaMut at pH 6.5 lacked tight tertiary packing, and as a result, the majority of residues were anticipated to be exposed to the bulk solvent. Therefore, it is reasonable to use the average chemical shifts of the exposed helix residues (Avbelj F., 2006) as a reference for comparisons. In this regard, by assuming a two-state (random coil and helix) folding model, the helix populations can be approximately represented by the ratio 100 between the chemical-shift deviations observed for different SH3 forms and those for the fully folded but exposed helix residues previously reported (Avbelj F., 2006). As seen in Figure4.4C, the distributions and percentages of helical conformations for the wild type at pH 2.0 and for4AlaMut at pH 6.5 are very similar, except that in 4AlaMut, the helix populations of residues close to the mutation sites were lower than those of corresponding residues in the wild type at pH 2.0. For the wild type at pH 2.0, ~70% of the residues showed >50% populated helical conformation, and more than half of the residues showed >70% populated helical conformation. In particular, the conformational shifts residues of Ser31, Trp35, and Arg36 were even higher than those calculated from the average chemical shifts expected for the exposed to solvent but fully folded helix. These results suggest that, except for the N-termini and C-termini, helical conformations are highly populated in both the wild type at pH 2.0 and 4AlaMut at pH 6.5. Consistent with the conformational chemical shift analysis, the NOE patterns defining secondary structures (Wagner, G., 1982) again support the proposition that helical conformations with a similar pattern were highly populated in the wild type at pH 2.0 and the 4AlaMut at pH 6.5. To be precise, according to the characteristic NOE connectivities in Figure4.5, such as dαN (i, i+2), dαN (i, i+3), dαN (i, i+4), and dNN (i, i+2), it shows that the Ntermini and C-termini were flexible without any restrains, whereas the remaining regions were transformed into two helices linked by a less structured loop over residues Leu28Asp29-Asp30 (in Figure4.6). However, due to the dominancy of dαN (i, i+2) NOEs and the rareness of dαN (i, i+4) NOEs, the helices observed here might be mainly 310-helices, which are rarely seen in native proteins but are regarded as a intermediate helical conformation existing in partially folded proteins. 101 Figure 4.4 Secondary chemical shifts and helix populations TOP: Bar plot of the difference between 13C Cα conformational shifts for the wild type at pH 6.5 (grey); wild type at pH 2.0 (black). MIDDLE: Bar plot of Hα conformational shifts for the wild type at pH 2.0 (black); 4AlaMut at pH 6.5 (grey). BOTTOM: The ratio (x 100) between Hα conformational shifts of the wild type at pH 2.0 (black); 4AlaMut at pH 6.5 (grey) and those of the fully folded but exposed helix residues (Avbelj, F. 2004). The secondary structure fragments are also indicated. 102 Figure 4.5 Characteristic NOEs defining secondary structures NOE connectivities identified for: a). Wild type at pH 2.0; b). 4AlaMut at pH 6.5. The plots are generated by CYANA 2.1. 103 Figure 4.6 Solution conformations Top: Ribbon representation of NMR structure of SH3 domain at pH 6.5. Left: Ribbon representation WT@pH2.0 with the lowest target function and its ensemble of ten structures superimposed over residues 32–44. Right: Ribbon representation of the NMR structure of the 4AlaMut SH3 domain at pH 6.5, with the lowest target function and its ensemble of ten structures superimposed over residues 32–44. 104 4.3.3 NMR 15N backbone relaxation The 15N NMR backbone relaxation data provide valuable information about the dynamics of the local environment of a protein on the picosecond-to-nanosecond timescale (Farrow, N. A., 1994; Mandel, A. M., 1995). In particular, the {1H}-15N heteronuclear steady-state NOE (hNOE) provides useful information of the backbone flexibility. So far in large number of SH3 domains, no regular secondary structure could be identified over the unique and long RT-loops, although they had extensive NOE connectivities with other parts of the molecules. Accordingly, the loop residues still have highly limited backbone motions and, consequently, relatively high hNOE values. As seen in Figure4.7A, except for the N-terminal residue Val1, the C-terminal residue Asn56, and Thr33 at the tip of the Src-loop, the NH vectors in wild type SH3 domain at pH 6.5 had values of hNOE >0.7 uniformly, with about half of them >0.8, indicating restricted bond motions in SH3 domain at neutral pH. However, some residues at the tips of the RT-loop (Trp7-Lys21) and Src-loop (Asp29-Arg36) yielded slightly lower hNOE values, indicating less limited backbone motions for these residues. By contrast, the hNOE values were significantly reduced for the wild type at pH 2.0 and for 4AlaMut at pH 6.5, suggesting less restricted backbone motions upon becoming partially folded. More specifically, the N-terminal and C-terminal residues had lower hNOE values for the wild type at pH 2.0 and 4AlaMut at pH 6.5, whereas the residues over two helices had higher hNOE values, with the majority >0.5 and some even >0.6, suggesting that the two helical fragments retained significantly limited backbone motions. Notably, the relatively small differences of hNOE values between helical and non-structured regions imply that non- 105 Figure 4.7 15N NMR backbone relaxation data 15N NMR backbone relaxation data for the wild type at pH 6.5 (yellow), wild type at pH 2.0 (blue) and 4AlaMut at pH 6.5 (red). (a) {1H}-15N steady-state NOE intensities, (b) 15N T1 (longitudinal) relaxation times, and (c) 15N T2 (transverse) relaxation times. All NMR experiments used for deriving these data were recorded at 278 K on an 800 MHz Bruker Avance NMR spectrometer. The mutation region is red-boxed and the secondary structure fragments are indicated. 106 structured regions are not random-coil with full freedom in backbone motions. Moreover, the T2 values also indicated similar patterns in which the residues located on two helical fragments had lower values than the N-terminal and C-terminal residues (Figure4.7C). In 4AlaMut at pH 6.5, the T2 values for the residues around the mutation region were larger than those of the corresponding residues in the wild type at pH 2.0. On the other hand, for both the wild type at pH 2.0 and 4AlaMut at pH 6.5, no uniform shortening of T2 values were observed, and the majority of their residues had T2 values larger than the corresponding residues of the wild type at pH 6.5, suggesting that no significant aggregation existed in the two helical states under the conditions used for the NMR investigations here. The reduced spectral density mapping of the wild type at pH2.0, the wild type at pH 6.5 and the 4AlaMut were calculated at three different frequencies: 0, 1H and 15 N, as shown in Figure 4.8. The J(0) and values differ for different parts of the proteins, in the same manner as relaxation parameters since J(0) is predominantly determined by R2, and by NOE. Except for a few residues, the spectral density of the 15N-1H vectors of the wild type SH3-1 at pH 6.5 has nearly uniform values: J(0) ≈ 2.5 ns/rad, J(ωN) ≈ ps/rad and ≈ 0.3 ns/rad. The wild type SH3-1 and 4AlaMut’s spectral density group into three obviously different parts, in terms of the J(0) and values. In the fragment (1-10aa), the J(0) value is around 1.5 ns/rad and is around 25 ps/rad; in the middle fragment (11-40aa), the J(0) value is around 2.0 ns/rad and 16 ps/rad; and in the third fragment (41-56aa) the J(0) value is around 1.0 ns/rad and 25 ps/rad. It is interesting to note that the first (N-terminal 1-10aa) and the last 107 Figure 4.8 Reduced spectral density mapping of wild type SH3-1 at pH2.0, at pH 6.5 and SH3-1 4AlaMut Calculations of spectral density function at 0, 1H and 15N frequencies, respectively. The black spots stand for the WT at pH 2.0; the red ones stand for the WT at pH 6.5; and the triangles stand for 4AlaMut. 108 fragment (C-terminal 41-65aa) are quite different from the middle one, in terms of their motion properties, and according to the spectral density at three different frequencies, which is almost consistent with the previous result that highly populated helical conformations exist in the two fragments Tyr9-Leu28 and Lys32-Thr44. On the other hand, faster motions contribute to the wild type at pH2.0 and 4AlaMut, since higher values are observed, as shown in Figure 4.8. 4.4 Discussion The folding study of β-lactoglobulin shows that a non-hierarchical mechanism might underlie the folding process and in this mechanism, a non-native helical intermediate is first formed from the collapse of a random coil, as driven by local interactions (Kuwajima, K., 1996; Hamada, D., 1996; Hamada, D., 1997; Arai, M., 1998; Fujiwara, K., 1999; Chikenji, G., 2000). The advantage of forming this kind of non-native helical intermediate is thought to be that it serves as a landmark to restrict the folding route to favour further transformations into the native β-sheet structure, which is difficult to access kinetically (Chikenji, G., 2000). Thus far, the non-native helical intermediate has been poorly documented and little information is available to clarify the folding pathway and kinetics of the β-sheet domains. In our study, the fact that the wild type form of the first hNck2 SH3 domain can be reversibly converted into a stable helical state at pH 2.0 offered an opportunity to investigate this kind of non-native helical states at equilibrium by multidimensional NMR 109 spectroscopy. The NMR parameters, such as chemical-shift deviations and NOE patterns, provided residue-specific evidence that the wild type SH3 domain did switch into a highly populated two-helix state at pH 2.0. In this state, the first and last β-strands became highly unstructured, whereas the RT-loop, together with the second β-strand, was converted into the N-helix, and the third and fourth β-strands switched into the C-helix. The 15 N NMR backbone relaxation data further indicate that despite being partially folded, the residues on the two helices retain significantly limited backbone motions. Interestingly, a previous NMR study identified a ~50% populated helical conformation formed over N-terminal residues 4–14 of the α-spectrin SH3 domain (Blanco, F. J., 1998). Here we speculate that the difference between the α-spectrin and Nck2 SH3 domains might mainly arise from their differential intrinsic properties for secondary-structure formation. One of the most challenging tasks in the non-hierarchical mechanism of β-protein folding is to understand what sequence determinants specify the second transition, in which local and long-range interactions must interplay properly to disrupt the helical conformation and simultaneously facilitate the formation of the final β-structure. To address this issue, we performed a bioinformatics study, and succeeded in identifying a four-residue region on the second strand which has a high intrinsic propensity to form a β-strand. Indeed, mutation of these residues to Ala causes the SH3 domain to form a helical state even at pH 6.5. Moreover, extensive NMR studies have demonstrated that the NMR conformational and dynamic properties of this mutant are highly similar to those of the wild type SH3 domain at pH 2.0. This observation has two implications: First, the 110 formation of the non-native helical state at pH 2.0 is unlikely to result from the specific effect imposed by the pH change. Instead, it is highly likely that acid unfolding nonspecifically destabilizes or disrupts the long-range interactions. Consequently, the intrinsic helix-formation propensity is released to drive the formation of the non-native helical conformation observed at pH 2.0. Second, the four-residue region Leu25-Trp26Leu27-Leu28 appears to play no significant role in the first transition, i.e., the formation of the helical state, because its mutation still allows the correct formation of the helical conformation, highly similar to that of the wild type at pH 2.0. However, these residues are essential for the second transition, to disrupt the helical conformation or to facilitate the formation of the final all-β SH3 fold by stabilizing the native β-structure. Moreover, our results highlight the complexity of the interplay between local and long-range interactions in specifying protein secondary and tertiary structures, consistent with a recent report that only a 12% sequence difference is able to switch a protein from a fullhelical structure to the α/β fold with completely distinctive secondary and tertiary structures (Alexander, P. A., 2007). Because the conformation of the two helical states determined here has no obvious similarity to the native all-β SH3 fold, it is challenging to speculate about the detailed mechanism underlying the second transition. However, examination of the native structure of the first hNck2 SH3 domain indicates that the four residues on the second β-strand have extensive long-range contacts with residues on other β-strands. A very interesting picture emerges with the inclusion of recently published results that mutating Ala45 to Gly would cause the Src SH3 domain to form a stable helical intermediate at pH 3.0 (Li, J., 2007). As seen in Figure 4.9, in both the hNck2 and Src SH3 domains, a tertiary hydrophobic core is formed among the side chains of the 111 residues corresponding to the four residues identified in our Nck2 SH3 domain and Ala45 found in the Src SH3 domain. This strongly implies that the establishment of tertiary packing might represent a key step in the second transition. The tertiary packing core may serve to disrupt the helical conformation in the non-native intermediate, and to direct the specific formation and assembly of the unique and complex SH3 fold. It would be of interest to identify the specific residues in the future to gain insight into detailed folding process. These results may also have implications in understanding protein misfolding-related diseases, such as prion disease and Alzheimer’s disease (Chiti, F., 2006; Ventura, S., 2004). For these diseases, the core mechanisms are associated with α-to-β conformation transitions. Previously it was found that for some protein fragments, one sequence is able to assume two distinctive secondary structures (hence termed the ‘‘chameleon sequence’’) (Guo, J.T., 2007). Given our observation that the uniquely defined all-β SH3 fold shared by more than 4000 sequences can highly populate a non-native helical conformation, it is likely that the existence of the chameleon sequences has more profound connotations than previously recognized. It’s consistent with the recent conclusion that most proteins, if not all, can be converted to amyloid fibers under proper conditions, although they adopt their native structures in physiological conditions. 112 Figure 4.9 Tertiary packing cores in the Src and first hNck2 SH3 domains a) Sequence alignment between the Src and first hNck2 SH3 domains. b) Tertiary packing cores in the first hNck2 SH3 domain formed by residues Leu25-Trp26-Leu27-Leu28 (yellow) identified in the present study and Val37 (red) corresponding to Ala45 reported (Li, J., 2007). c) Tertiary packing cores in the Src SH3 domain formed by residues Ala45 (red) and Leu32-Gln33-Ile34-Val35 (yellow) corresponding to Leu25-Trp26-Leu27-Leu28. d) SH3 contact area plot. The marked one row and two columns are residues L27, L25 and V37. The box is contact area L25-L28 with V37. 113 4.5 Conclusion The studies of several proteins implied that the folding of β-proteins may follow a nonhierarchical mechanism in which two major transitions are essential, i.e., the collapse of a random coil to form a non-native helical intermediate, followed by a transformation into the native β-structure. The first hNck2 SH3 domain which adopted an all-β barrel in the native form can be reversibly transformed into a stable and non-native helical state by acid-unfolding. The extensive NMR and mutagenesis studies led to two striking findings: First, NMR analysis reveals that in the helical state formed at pH 2.0, the first and last βstrands in the native form become unstructured, whereas the rest is surprisingly converted into two highly populated helices with a significantly limited backbone motion; Second, a conserved four-residue sequence is identified on the second β-strand, a mutation of which suddenly renders the SH3 domain into a helical state even at pH 6.5, with NMR conformational and dynamic properties highly similar to those of the wild type at pH 2.0. This observation implies that the region might contribute key interactions to disrupt the helical state, and to facilitate a further transformation into the native SH3 fold in the second transition. 114 [...]... superimposed over residues 32 44 Right: Ribbon representation of the NMR structure of the 4AlaMut SH3 domain at pH 6.5, with the lowest target function and its ensemble of ten structures superimposed over residues 32 44 1 04 4.3.3 NMR 15N backbone relaxation The 15N NMR backbone relaxation data provide valuable information about the dynamics of the local environment of a protein on the picosecond-to-nanosecond... Tyr9-Leu28 and Lys32-Thr 44 On the other hand, faster motions contribute to the wild type at pH2.0 and 4AlaMut, since higher values are observed, as shown in Figure 4. 8 4. 4 Discussion The folding study of β-lactoglobulin shows that a non-hierarchical mechanism might underlie the folding process and in this mechanism, a non-native helical intermediate is first formed from the collapse of a random... our Nck2 SH3 domain and Ala45 found in the Src SH3 domain This strongly implies that the establishment of tertiary packing might represent a key step in the second transition The tertiary packing core may serve to disrupt the helical conformation in the non-native intermediate, and to direct the specific formation and assembly of the unique and complex SH3 fold It would be of interest to identify the. .. although they adopt their native structures in physiological conditions 112 Figure 4. 9 Tertiary packing cores in the Src and first hNck2 SH3 domains a) Sequence alignment between the Src and first hNck2 SH3 domains b) Tertiary packing cores in the first hNck2 SH3 domain formed by residues Leu25-Trp26-Leu27-Leu28 (yellow) identified in the present study and Val37 (red) corresponding to Ala45 reported... that the wild type SH3 domain did switch into a highly populated two-helix state at pH 2.0 In this state, the first and last β-strands became highly unstructured, whereas the RT-loop, together with the second β-strand, was converted into the N-helix, and the third and fourth β-strands switched into the C-helix The 15 N NMR backbone relaxation data further indicate that despite being partially folded, the. .. secondary and tertiary structures (Alexander, P A., 2007) Because the conformation of the two helical states determined here has no obvious similarity to the native all-β SH3 fold, it is challenging to speculate about the detailed mechanism underlying the second transition However, examination of the native structure of the first hNck2 SH3 domain indicates that the four residues on the second β-strand have... ns/rad and 16 ps/rad; and in the third fragment (41 -56aa) the J(0) value is around 1.0 ns/rad and 25 ps/rad It is interesting to note that the first (N-terminal 1-10aa) and the last 107 Figure 4. 8 Reduced spectral density mapping of wild type SH3- 1 at pH2.0, at pH 6.5 and SH3- 1 4AlaMut Calculations of spectral density function at 0, 1H and 15N frequencies, respectively The black spots stand... type at pH 2.0 On the other hand, for both the wild type at pH 2.0 and 4AlaMut at pH 6.5, no uniform shortening of T2 values were observed, and the majority of their residues had T2 values larger than the corresponding residues of the wild type at pH 6.5, suggesting that no significant aggregation existed in the two helical states under the conditions used for the NMR investigations here The reduced spectral.. .the range from 5 to 100 mM for the wild type and 4AlaMut Superimposition of the spectra showed no obvious difference, indicating they were concentration-independent and no significant aggregation occurs under these concentrations 4. 3.2 NMR characterization and modelling Isotope-labelled recombinant wild type hNck2 SH3- 1 and 4AlaMut SH3- 1 were also prepared for a detailed characterization by NMR. .. clarify the folding pathway and kinetics of the β-sheet domains In our study, the fact that the wild type form of the first hNck2 SH3 domain can be reversibly converted into a stable helical state at pH 2.0 offered an opportunity to investigate this kind of non-native helical states at equilibrium by multidimensional NMR 109 spectroscopy The NMR parameters, such as chemical-shift deviations and NOE patterns, . 2001; Ran, X., 2005). The NMR structure of the first Nck2 SH3 domain was previously determined (Park S, 2006), and the structures of the second and third SH3 domains as well as the SH2 domain were. representation of the NMR structure of the 4AlaMut SH3 domain at pH 6.5, with the lowest target function and its ensemble of ten structures superimposed over residues 32 44 . 105 4. 3.3 NMR 15 N. β-stands, which are organized into two β-sheets. The first, the last and the first half of the second β-strand constitute the first β-sheet, whereas the second half of the second β-strand,