Chapter Folding and amyloidogenesis study of hNck2 SH3-1 domain 115 5.1 Introduction The aggregation and deposition of biomolecules are believed to be underlying causes of many human diseases, including atherosclerosis, amyloidosis, gallbladder and kidney stones. Amyloidosis, characterised by the deposition of abnormal protein fibres in various tissues and organs, is thought to be responsible for critical diseases like Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease, progressive muscle atrophy, and prion disease. Under appropriate conditions, most proteins examined seem capable of fibre formation in vitro, leading to the belief that this process is a generic property of polypeptides (Fandrich M, 2001). In vivo, restrained physiological conditions confine the number of proteins capable of amyloid fibre formation to a few dozen, all of which, unfortunately, lead to some kind of other disease (Tan SY, 1994). Amyloid fibre formation occurs both intra- and extracellular and fibres are often co-aggregated with lipid membranes and calcified (Kidd M, 1963; Kidd M, 1964). The Src-homology region 3(SH3) domains are well characterised, and small protein modules of 60–85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The folding and unfolding of this small domain is an apparent two-state reaction under most conditions investigated so far. In spite of the apparent two-state character of its folding, NMR-detected hydrogendeuterium exchange experiments (Sadqi, M., 2002; Sadqi, M., 1999) have indicated that under native conditions, the Spc-SH3 domain undergoes a wide variety of conformational fluctuations, ranging from local motions to extensive structural disruptions affecting its core. Oligomerisation of partially unfolded intermediates has been proposed to be playing a crucial role in the initial stages of amyloid formation, by further evolving into the formation of larger aggregates, protofibrils and finally 116 amyloid fibrils (Dobson, C. M., 2003). In this study, the malignant hNck2 SH3-1 domain will be expressed in our system to investigate the structure properties and its amyloidogenesis. 5.2 Materials and methods 5.2.1 Clone and mutagenesis The V22 insertion was obtained by PCR-based de novo design according to the AAC04831 NCBI published protein sequence. The hNck2 SH3-1 mutation constructs, including V22K, V22D and V22A, were obtained by mutagenesis. The primers used in the mutagenesis are listed below: V22D_f: 5’-GGAACTGGATATTAAAAAAGATAACGAACGTCTGTGGC-3’ V22D_r: 5’-GCCACAGACGTTCGTTATCTTTTTTAATATCCAGTTCC-3’ V22K_f: 5’-GGAACTGGATATTAAAAAAAAAAACGAACGTCTGTGGC-3’ V22K_r: 5’-GCCACAGACGTTCGTTTTTTTTTTTAATATCCAGTTCC-3’ V22A_f: 5’-GGAACTGGATATTAAAAAAGCGAACGAACGTCTGTGGC-3’ V22A_r: 5’-GCCACAGACGTTCGTTCGCT TTTTTAATATCCAGTTCC-3’ 5.2.2 Protein expression and purification The proteins (unlabelled and 15 N labeled) were expressed as described in chapter 4. SH3-1-V22, -D22, -A22, -K22 and 4AlaMut were expressed as the inclusion body component and purified under the denatured condition. The wild type SH3-1 were 117 expressed as supernatant components in the BL21 strain and purified under native conditions. All proteins were further purified by HPLC. 5.2.3 CD and NMR spectra of mutant SH3 domain For the wild type SH3-1 protein, the CD and NMR samples were prepared by bufferexchange with mM phosphate buffer at pH 6.5. For SH3-1-V22, -A22, -D22 and K22 mutants, the lyophilised protein powder was dissolved in the deionised water (Millipore, Milli-Q) with the addition of 10% D2O in the NMR samples for spin-lock. Since both the high salt concentration buffer and the pH will dramatically affect the solubility of these proteins, most CD and NMR experiments were performed with a mM salt concentration at pH 4.0. However, the CD and NMR HSQC experiments of the SH3-1-V22 mutant were collected at both pH 4.0 and 6.5 for comparison. CD experiments were performed on a Jasco J-810 spectropolarimeter equipped with a thermal controller as described in 2.3. The far-UV CD spectra were collected at a peptide concentration of ~50 μM at 25 ºC . The near-UV CD spectra were collected at a protein concentration of ~200 μM in the absence and in the presence of M urea. Five independent scans were collected and the averaged ones were recorded for further analysis. NMR experiments were acquired on an 800 MHz Bruker Avance Spectrometer equipped with pulse field gradient units at 298 K. The NMR spectra acquired for both the backbone and side chain assignments included the 15 N-edited HSQC-TOCSY, HSQC-NOESY and the 13C-HCCH-TOCSY, as well as triple-resonance experiments (HNCACB, CBCA(CO)NH, HNCO). NOE connectivity was identified from the 15N- 118 NOESY spectrum. 15 N T1 and T2 relaxation times and {1H}-15N steady-state NOEs experiments were performed on the 800-MHz Spectrometer at 298 K. 15 N T1 relaxation experiments were conducted with delays of 10, 100, 200, 300, 400, 500, 600 and 700 msec. 15N T2 ones had delays of 10, 60, 100, 130, 160, 200, 230, 260 and 300 msec. {1H}–15N steady-state NOEs were obtained by acquiring spectra with and without H presaturation of a duration of three seconds and a relaxation delay of five seconds at 800 MHz. NMR data were first processed with an NMRPipe and subsequently analysed and fitted by NMRView. The solution structure of the Nck2 SH3-1 (2B86) was obtained from PDB and its NMR assignment with an accession code of 6854 was downloaded from BioMagResBank. The graphic software MolMol was used for structure display and analysis. 5.2.4 Amyloidogenesis condition screening and EM samples preparation The SH3-1-V22, the 4AlaMut and the wild type SH3-1 at pH3.0 were investigated for their amyloid formations. Protein concentration was fixed at 3mg/ml, while a series of sodium chloride concentrations: 10, 20, 50, 100, 200, 300 and 500 mM were chosen for the optimisation of amyloid formation. The results were examined by TEM after seven days of incubation. EM grids were processed by the Drop-To-Drop method, as follows. (1) 5μl of liquid specimen was placed on the parafilm; (2) The carbon-coated 100-mesh copper grid was placed on the drop and allowed to absorb for approximately one minute; (3) A filter paper was used to wick away specimen drops; (4) A grid was placed on the drop of filtered 1% phosphotungstic acid (PTA) at pH 3.0, and allowed to stain for 30-60 seconds; (5) The excess fluid was wicked away 119 with filter paper, and the grids were placed with specimen-side up. Photographs were taken from a JEOL JEM2010F Electron Microscopy equipped with CCD. Magnifications were chosen as 8000× and 20000×. 5.3 Results 5.3.1 Biological activity of wild type SH3-1 Initially the Nck2 SH3-1 was cloned as malignant form in which an additional Val was inserted at the tip of diverging turn. Subsequent protein expressions showed that the SH3-1-V22 went into the inclusion body portion. Accordingly, the additional Val residue was removed to testify whether the native tertiary structure can be restored. And to test whether wild type SH3-1 still has any biological activity, the CD3ε peptide, which was previously shown to be capable of binding the Nck2 SH3-1 was also expressed as a GST-fusion protein. After cleavage, purification and lyophilisation, the HSQC perturbation experiment was performed to verify the binding result. Significant HSQC peak shifts were observed for the wild type in the buffer condition (50mM phosphate, pH 6.5), while no chemical shifts were detected for the four insoluble SH3-1 domains in salt-free water, indicating that the extra presence of residue at a diverging turn will abolish the structure’s compactness and biological binding activity. 5.3.2 Mutagenesis study of SH3-1-V22 To clarify whether different types of residue at the same position of Val have the same deteriorating effect, three other residues with distinguished side-chain properties were introduced by mutagenesis; namely, SH3-1-K22, -A22, and -D22. The 15 N 120 labelled samples were prepared as described before. HSQC spectra of these mutants which were dissolved in pure water were acquired as shown in Figure 5.1 A-D. The SH3-1-V22 HSQC spectrum at pH2.0 and pH 4.0 are superimposed in Figure 5.1 F. For comparison, the wild type SH3-1, which was dissolved in the phosphate buffer at pH 6.5, is also illustrated here. A narrow dispersion ~1 ppm at 1H dimension and 18 ppm at 15 N dimension indicate that SH3-1-V22, -A22, -K22 and -D22 are in an unstructured state. Meanwhile, these mutants and the wild type SH3-1 were also studied by CD, as shown in Figure 5.2. The mutants were all dissolved in pure water and far-UV spectra were acquired from 260nm to 190nm. The wild type SH3-1 dissolved in the buffer condition was also acquired. All mutants show unstructured states according to the CD profiles, while the wild type SH3-1 shows a β sheet dominant structure. From both CD and NMR preliminary studies, it showed that regardless of the residue types at the tip of a diverging turn for all mutants, it would prevent the SH3-1 domain from achieving a well folded native structure. 121 Figure 5.1 HSQC of mutants and refolded WT SH3-1 The SH3-1-A22, -D22, -K22 and V22 that are dissolved in pure water HSQC spectra are shown in Figures A-D. V22del (buffer, pH6.5) HSQC is illustrated in Figure E. SH3-1-V22 HSQC spectra at pH2.0 and pH 4.0 are compared in Figure F. 122 Figure 5.2 CD Comparison of mutants and WT SH3-1-A22, -D22, -K22, -V22 and wild type CD spectra are compared with each other. Different colours are designated to different mutants and WT, as is shown in the right-hand corner. 123 5.3.3 Assignment, secondary CS analysis and dynamics study of SH3-1-V22 As mentioned in the previous section, the insertion at the tip of the diverging turn of the SH3-1 significantly changes the structural properties and in addition, one residue at this critical point retains the protein in a denatured state, according to CD and NMR studies. It is fundamentally interesting to investigate the structural and dynamics properties of such an insoluble protein. The double-labeled SH3-1-V22 sample was first prepared to conduct a sequential assignment. The assigned HSQC spectrum of the SH3-1-V22 and the V22△ at 8M urea are illustrated in Figure 5.3. To reflect the real denatured situation, the V22△ was dissolved in 8M urea and the assignment was done to provide a reference for SH3-1-V22 chemical shift. The secondary chemical shifts of the SH3-1-V22 for 1Hα, 13Cα, 13Cβ and 13CO were successfully obtained. To analyse the secondary chemical shifts of SH3-1-V22 and the tendency to form a secondary structure element, as well as the denatured status after the introduction of additional residue at the tip of a diverging turn, the averaged helix/strand chemical shifts both in buried and exposed states that are obtained from Franc Avbelj (Franc Avbelj, 2004) are used for comparison. As shown in Figure 5.4, the proton and carbon secondary chemical shifts of the SH3-1-V22 at pH 4.0 and WT SH3-1 at pH 6.5 from the Wagner Group were compared (Figure 5.4 A-B). The proton and carbon secondary chemical shifts of SH3-1-V22 are also compared with those of WT SH3-1 at pH 2.0 (Figure 5.4 C-D). The alpha proton secondary chemical shifts of SH3-1-V22 are compared with those of helix at both buried and exposed states (Figure 5.4 E-F). In the cases of the buried and exposed helix, high similarities are observed for some residues with an exposed secondary chemical shifts that are closer to the secondary chemical shift of the SH3-1-V22. From both the proton and carbon secondary 124 5.3.4 Identification of non-native medium-range NOEs and native-like longrange NOEs To gain an insight into structural and packing properties, we have acquired both the 15 N- and 13 C-edited NOESY spectra of the denatured SH3-1-V22. An NOE analysis shows to some extent the persistence of the tertiary contacts. Unfortunately, due to a severe signal overlap arising from resonance degeneration, it was impossible to assign the 13C-NOESY spectrum. Nevertheless, the NOEs from the 15 N-NOESY spectrum can still be assigned due to the good dispersion in the 1H and 15 N dimensions of the spectra. Interestingly, in addition to intra-residual ones, sequential and medium-range NOEs can be observed for almost all residues. In particular, 23 residues (~40% of the molecule) still owned long-range NOEs. A detailed comparison of the persistent NOEs in the denatured SH3-1-V22 with those of the published native Nck2 SH3-1 (Park S, 2006) led to a classification of the remaining NOEs into nonnative and native-like categories (Tables 5.1 and 5.2). A close examination of the nonnative NOEs revealed that they were all sequential and medium-range NOEs located almost all over the molecule. As shown in Table 5.1, many sequential NH-NH and α/βH(i)-NH(i+2) NOEs that are characteristic of the loop/turn/nascent-helix conformations suddenly show up on five β-strands. These NOEs were totally inconsistent with well-formed and rigid β-strands in the published SH3-1 structure (Park S, 2006), thus clearly indicating that in the denatured SH3-1-V22, th e fiv e βstands became distorted, or/and lost its rigidly extended backbone conformation. Similarly, non-native NOE patterns were also observed in the two RT-loop strands. On the other hand, many native-like medium-range NOEs over the β-turn/loop regions could be identified. Specifically, except for the absence of long-range NOEs 130 on the fifth β-strand, the β-strands 1–4 and the two RT-loops all were characterised by native-like long-range NOEs (Table 5.2). For example, two native-like long-range NOEs were found between the first and second β-strands, six between the second and third strands, seven between the third and fourth strands, and seven between the two RT-loop strands. No long-range NOEs in the fifth β-strand were detected, probably because of the loose packing in the rest of the molecule. 131 Table 5.1 Non-native medium range NOEs Loop and two RT-loop strands Trp7 HN Val3 HB Trp7 HN Val3 HG Trp7 HN Ile4 HG Trp7 HN Ala5 HB Trp7 HN Ala5 HA Asp8 HN Ala5 HB Asp8 HN Lys6 HB Asp8 HN Lys6 HE Asp8 HN Ala11 HB Thr10 HN Trp7 HA Thr10 HN Trp7 HB Ala11 HN Trp7 HA Thr10 HN Gln12 HB Thr10 HN Gln12 HG Lys20 HN Val22 HN Val22 HN Glu24 HA Val22 HN Lys20 HB Val22 HN Lys20 HG Val22 HN Lys20 HA Val22 HN Lys20 HD Asn23 HN Arg25 HG The first beta-strand Val3 HN Ile4 HN Ala5 HN Val3 HA Ala5 HN Val3 HB The second beta-strand Leu26 HN Glu24 HB Leu26 HN Glu24 HA Leu29 HN Trp27 HA Asp31 HN Leu29 HA Asp31 HN Leu29 HB The third beta-strand Arg37 HN Val38 HN Arg37 HN Trp36 HN Asn40 HN Val38 HA Asn40 HN Val38 HB Arg37 HN Arg39 HA The fourth beta-strand Tyr47 HN Gly46 HN Tyr47 HN Thr45 HG The fifth beta-strand Tyr52 HN Glu54 HG Val53 HN Tyr52 HN Glu54 HN Arg55 HN Glu54 HN Val53 HN Glu54 HN Tyr52 HB Arg55 HN Val53 HG Arg55 HN Val53 HB Table 5.2 Native like long range NOEs Between the first and second beta strands Val3 HN Leu26 HB Val3 HA Trp27 HE Between the second and third beta strands Arg25 HN Asn40 HB Trp27 HE Asn40 HD Trp27 HE Asn40 HB Trp27 HE Arg39 HB Leu29 HN Arg39 HE Trp35 HE Asp31 HA Between the third and fourth beta strands Arg39 HN Thr45 HA Asn40 HN Thr45 HA Arg44 HH Arg39 HB Tyr47 HN Arg37 HA Tyr47 HN Trp36 HB Tyr47 HN Val38 HB Val48 HN Trp35 HA Between the RT loop and fourth beta strand Leu17 HN Thr45 HA Arg44 HH Leu17 HD Between the two RT loop strands Lys20 HN Trp7 HA Lys20 HN Trp7 HB Trp7 HE Lys20 HG Tyr9 HN Ile19 HA Tyr9 HN Asp18 HB Tyr9 HN Asp18 HB Tyr9 HN Ile19 HB 132 5.3.5 Salt effect on SH3-1-V22 To obtain useful information of SH3-1-V22 in the partially folded state, after lyophilisation, the protein was dissolved in pure water to prevent aggregation and precipitation. At the same time, the same experiment except for additional salt in solution was also conducted to investigate whether the salt had any effect on SH3-1V22’s stability and solubility. If proteins would aggregate or precipitate, big differences would be observed in terms of line-width and the intensity of cross-peaks. As shown in Figure 5.6, the 1D spectra of salt-free samples were collected. In comparison, the 1D spectra immediate, one hour and one week after adding 100mM NaCl salt were also acquired. Severe line-broadening and decreased intensities were observed even after the one week incubation, indicating the oligomerization of SH31-V22 and/or the change of dynamic properties and a still low population of SH3-1V22 in the monomeric state. Interestingly, the fact that higher concentrations of NaCl salt will lead to SH3-1-V22 precipitation gives rise to the question of what the effect of ion strength would be on the unfolded protein. 133 Figure 5.7 Comparison of 1D NMR spectra at different elapsed times after adding 100mM salt A) shows the water-free spectrum of SH3-1-V22. B, E and F show the 1D spectra after immediate, one hour and one week. C, D, G and H show the zooming of A, B, E and F, respectively. All acquisition parameters are the same for the comparison. 134 5.3.6 Amyloidogenesis study of SH3-1-V22, mutants and WT SH3-1 The amyloidogenesis has been intensively studied in recent years. The denatured condition usually gives rise to the unfolding of a protein and the beginning of the formation of amyloid fibrils. In this study, the formation of fibril was studied in a wide range of salt concentration from 5mM to 500mM. The incubation lasted for one week and the temperature was kept constantly at 25℃. The samples preparation is described in the Materials and Methods section. As shown in Figure 5.7, the amyloid fibrils were observed at salt concentrations of 20mM, 50mM and 200mM. At other salt conditions, either no amyloid was observed or a high irregular precipitation was observed. 135 Figure 5.8 SH3-1-V22 amyloidogenesis at different salt concentrations A–C), the NaCl concentration is 200mM, with A and B, 8000 magnification, and C, 20000 magnification. D– G) The NaCl concentration is 20 mM, with D and F 8000×, E and G 20000×. H–I) The salt concentration is 50 mM, with H 8000× and I 20000×. All pictures are taken from a JEOL JEM2010F Electron Microscopy equipped with CCD. 136 Figure 5.9 EM pictures of 4AlaMut at pH3.0/pH6.5 A–B) 4AlaMut, at pH3.0, 150mM NaCl. C–D) 4AlaMut at pH 6.5, 10mM NaCl. The magnifications are shown in each figure (bottom-left corner). 137 Figure 5.10 EM pictures of WT at pH3.0 A–F) WT at pH3.0, 150mM NaCl. The magnifications are shown in each figure (bottom-left corner). 138 Figure 5.11 EM pictures of WT at pH6.5 A–F) Wild type at pH6.5, 150mM NaCl. The magnifications are shown in each figure (bottom-left corner). 139 5.4 Discussion 5.4.1 Insertion at tip of diverging turn affects protein folding, solubility and stability of SH3-1 domain At present, understanding the relationship between protein folding, protein solubility and stability still represents a challenge. The well folded protein structure can be solved by X-ray crystallography and the NMR method. For the partially folded protein, due to the equilibrium between the conformers and highly dynamic backbone movement, it is impossible to solve the high resolution structure of the protein in a denatured condition. NMR technique, on the other hand, can provide us enriched information of partially-folded protein, allowing us to investigate its folding properties and structure-function relationship. Among these, the SH3 protein has been well studied as an ideal folding model, due to its small size (50–70) and the absence of disulphide bonds. In our study, the template AAC04831 we initially chose encoded an extra residue Val at the tip of diverging turn, which was highly conserved as Gly in the SH3 family. This accidentally selected template led to the expressions as the inclusion body components for all proteins carrying this residue. The deletion of this residue resulted in an expression as the supernatant component, and the refolding process was very successful, when the pH was gradually changed to 6.5. It was also certain that the deteriorating effect caused by the insertion was not residue-dependent, because all mutants (SH3-1-V22, -A22, -K22 and -D22) were characterised by a narrow spectral dispersion. A recent mutation study showed that the T22G of the drkN SH3 domain completely stabilised the protein energetically (Irina Bezsonova, 2005). This result indicated that the Gly was much conserved, and any mutation at this point would 140 energetically affect the folding process of the SH3 domain which will probably lead to its low solubility and stability eventually. In our study, the peptide I1K2K3N4E5R6L7 of wild type SH3-1 will normally adopt typical diverging β-turn structure in isolation based on the previous observation (Bystroff and Baker, 1998). The first contact is the hydrogen bond between the carbonyl oxygen of K3 and the amide proton of E5. The second contact is the mainchain-sidechain hydrogen bonding between the amide proton of K2 and the sidechain carboxylate oxygen of E5. The final contact is the hydrophobic sidechain contact. Out of these three local contacts, the first one is the most important, showing up with the highest frequency in most of type II β-turn structures. It can be speculated that the N22 of the wild type may reduce the stability of the SH3-1, according to the mutation study of T22G (Irina Bezsonova, 2005). However, the study of insertion at the tip diverging turn following the second K has not been deliberated until now. After the insertion of Val, the local contacts between K and E are spatially contorted, due to the increased space between them. Without the first and second contacts in the β-turn, it can be expected that it is extremely difficult for the peptide IKKVNERL to remain a typical structure of the type II β-turn. Therefore, in the absence of the correctly folded type II β-turn, a further folding of the typical SH3 domain is hampered. 5.4.2 Reduced population of helical conformation and native-like long range NOEs of SH3-1-V22 suggest the intermediate state between highly populated helical conformation and natively folded state The characterisation of protein folding should not only include a description of the well folded structure, but also the denatured state, which is an ensemble of many 141 different conformations in rapid exchange. NMR Spectroscopy has become an extremely useful tool for the detailed structural characterisation of the denatured states of proteins, and it is made possible by the development of multidimensional methods using isotopically enriched samples (Wuthrich, 1994). In this study, comparisons of SH3-1-V22 secondary chemical shifts with the buried and exposed ones showed a high similarity with exposed secondary chemical shifts. The comparison of SH3-1-v22 with WT at pH2.0 showed a reduced tendency to form a helical conformation. Interestingly, non-native NOEs and native-like long range NOEs were observed at the same time, indicating a reduced helical tendency and an intermediate state approaching the native fold. The reduced helical tendency may result from an insertion at the tip of diverging turn and the raised pH value to 4. From the secondary chemical shift comparison between the wild type SH3-1 at pH2.0 and SH3-1-V22, the latter shows a reduced value, especially in the region around the V22. This can be interpreted as a reduced population of the helical conformation in fast exchange with the highly populated random-coil conformation. It is difficult to expect which factors (insertion or pH change) cause the less populated helical conformation or a combination of the two, because of the limited detection methods. The hetNOE and dynamics relaxation parameters were also obtained for the SH3-1V22. To examine the suspicious similarity between the WT at pH2.0 and the SH3-1V22 state, the hetNoe and relaxation parameters were compared. The hetNOE values of the SH3-1-V22 are smaller than the ones of the WT at pH2.0, except for the β3 strand region and the region between β2 and β3, indicating that the SH3-1-V22 is more flexible than the WT at pH2.0, which is also consistent with the result that the WT at pH 2.0 has a more populated helical conformation. The T2 values of SH3-1- 142 V22 are homogeneously larger than those of the wild type at pH2.0, indicating less rigidness in the SH3-1-V22, as compared with the WT at pH 2.0. In this study, the secondary chemical shift, NOEs and dynamics of the SH3-1-V22 were analysed and compared with those of the wild type at pH2.0. The reduced conformational chemical shifts, the existence of non-native like medium-range NOEs and native-like long-range NOEs at the same time, and a more flexible property reflected in dynamic analysis, indicate that the SH3-1-V22 has a reduced tendency to form a helical conformation and a lower population of helical conformations that fast exchange with the random-coil state. A number of native-like, long-range NOEs show that the SH3-1-V22 might be an intermediate state that is much closer to the native fold. 5.4.3 Aggregation, amyloidogenesis and precipitation of SH3-1-V22 In the beginning of the design and expression of SH3-1-V22, its sequence was accidentally picked up from early deposited sequence that was shown an additional residue in the first SH3 domain and the protein encoding this fragment was expressed as the inclusion body component. Precipitations in the buffer condition were observed for all mutants that had the extra inserted residue at the tip of diverging turn of SH3-1. A sequence analysis showed that the insertion at this site spatially distorted the local contacts between K and E in the diverging turn. As a result, lack of proper initial folding of type II β-turn structure leads to intermediate conformation of SH3-1 between highly populated helical conformation and natively folded SH3 domain, or possibly between the random coil conformation and helical conformation. 143 The NMR 1D spectra obtained in the presence of 100 mM of NaCl showed a linebroadening after one hour, indicating the increasing salt concentration promotes oligomerisation of SH3-1. The 2D HSQC were also acquired, even one week after incubation. Although the peak intensity declined steeply, acquisition sensitivity was still high enough to record 2D spectra, indicating that the residue portion of SH3-1V22 still existed as monomeric or oligermeric forms. As we had noted previously, the SH3-1-V22 was less constrained, compared with the wild type at pH2.0, and the exposed hydrophobic residues played a crucial role in the early stage of amyloid formation by further evolving into the larger aggregates, protofibrils and amyloid fibrils. The fact that 4AlaMuat would also form protofibrils and amyloid fibrils indicates similar propensity of SH3-1 to form fibrils under denatured conditions even with changed residue in the middle region. 5.5 Conclusion In this chapter, systematic studies by different methods were conducted on the SH3-1V22, which had one extra non-native Val residue insertion at the tip of diverging turn. The insertion at this critical point spatially distorted the local contacts of the diverging turn. With a loss of the critical mainchain-mainchain hydrogen bond and mainchainsidechain hydrogen bond between K and E in the diverging turn, the early folding was disrupted after the insertion of Val, even in the first coil-helix transition, exhibiting lower tendencies to form helical confirmations, as compared with the wild type SH3-1 at pH2.0. The reduced conformational chemical shifts of SH3-1-V22 showed a 144 reduced population of helical conformations that fast exchanged with the random-coil conformations. The coexistence of non-native NOEs and native-like long-range NOEs imply the reduced helical tendency and the intermediate state which is close to the native fold or in the helix-coil transition. The oligomerisation of SH3-1-V22 in the presence of 100 mM of NaCl salt might be an initial stage to form protofibrils and finally amyloid fibrils. The 4AlaMut and wild type SH3-1 form amyloid fibrils at both a neutral and an acidic pH, indicating that under certain conditions, proteins are capable of forming amyloid fibril. 145 [...]... are the same for the comparison 134 5. 3.6 Amyloidogenesis study of SH3- 1-V22, mutants and WT SH3- 1 The amyloidogenesis has been intensively studied in recent years The denatured condition usually gives rise to the unfolding of a protein and the beginning of the formation of amyloid fibrils In this study, the formation of fibril was studied in a wide range of salt concentration from 5mM to 50 0mM The. .. Figure 5. 5 A reduced spectral density mapping was calculated at 0, 1H and 15N frequencies Big differences can be observed between the SH3- 1-V22 and WT at 0 and 1H frequencies, especially at the N- and C-terminals of SH3- 1 1 25 Figure 5. 3 Assignments of SH3- 1-V22 at pH 4.0 and V22△ in 8M urea The upper panel is the SH3- 1-V22 assignment and the lower panel is the V22△ assignment The stars stand for the his-tag... for the SH3- 1V22 To examine the suspicious similarity between the WT at pH2.0 and the SH3- 1V22 state, the hetNoe and relaxation parameters were compared The hetNOE values of the SH3- 1-V22 are smaller than the ones of the WT at pH2.0, except for the β3 strand region and the region between β2 and β3, indicating that the SH3- 1-V22 is more flexible than the WT at pH2.0, which is also consistent with the. .. contact is the hydrogen bond between the carbonyl oxygen of K3 and the amide proton of E5 The second contact is the mainchain-sidechain hydrogen bonding between the amide proton of K2 and the sidechain carboxylate oxygen of E5 The final contact is the hydrophobic sidechain contact Out of these three local contacts, the first one is the most important, showing up with the highest frequency in most of type... Val53 HN Glu54 HN Tyr52 HB Arg 55 HN Val53 HG Arg 55 HN Val53 HB Table 5. 2 Native like long range NOEs Between the first and second beta strands Val3 HN Leu26 HB Val3 HA Trp27 HE Between the second and third beta strands Arg 25 HN Asn40 HB Trp27 HE Asn40 HD Trp27 HE Asn40 HB Trp27 HE Arg39 HB Leu29 HN Arg39 HE Trp 35 HE Asp31 HA Between the third and fourth beta strands Arg39 HN Thr 45 HA Asn40 HN Thr 45. .. SH3- 1-V22, the 15N NMR backbone relaxation data of the SH3- 1-V22 with a protein concentration of ~700 μM were collected in salt-free water (pH 4.0) at 25 on an 800 MHz Bruker Avance NMR Spectrometer As shown in Figure 5. 5, the HetNOE, T1 and T2 parameters are presented in figures A, B and C sequentially The relaxation parameters of the wild type SH3- 1 are also presented and compared with those of the SH3- 1-V22,... type II β-turn structures It can be speculated that the N22 of the wild type may reduce the stability of the SH3- 1, according to the mutation study of T22G (Irina Bezsonova, 20 05) However, the study of insertion at the tip diverging turn following the second K has not been deliberated until now After the insertion of Val, the local contacts between K and E are spatially contorted, due to the increased... between them Without the first and second contacts in the β-turn, it can be expected that it is extremely difficult for the peptide IKKVNERL to remain a typical structure of the type II β-turn Therefore, in the absence of the correctly folded type II β-turn, a further folding of the typical SH3 domain is hampered 5. 4.2 Reduced population of helical conformation and native-like long range NOEs of SH3- 1-V22... (Table 5. 2) For example, two native-like long-range NOEs were found between the first and second β-strands, six between the second and third strands, seven between the third and fourth strands, and seven between the two RT-loop strands No long-range NOEs in the fifth β-strand were detected, probably because of the loose packing in the rest of the molecule 131 Table 5. 1 Non-native medium range NOEs Loop and. .. density calculated at 0, 1H and 15N frequencies The closed circle stands for SH31 -V22 at pH 4.0 and the open circle stands for the wild type SH3- 1 at pH 6 .5 129 5. 3.4 Identification of non-native medium-range NOEs and native-like longrange NOEs To gain an insight into structural and packing properties, we have acquired both the 15 N- and 13 C-edited NOESY spectra of the denatured SH3- 1-V22 An NOE analysis . beta-strand Tyr52 HN Glu54 HG Val53 HN Tyr52 HN Glu54 HN Arg 55 HN Glu54 HN Val53 HN Glu54 HN Tyr52 HB Arg 55 HN Val53 HG Arg 55 HN Val53 HB Between the first and second beta strands Val3. mapping of SH3- 1-V22 and WT SH3- 1 A, B and C are spectral density calculated at 0, 1 H and 15 N frequencies. The closed circle stands for SH3- 1-V22 at pH 4.0 and the open circle stands for the. fitted by NMRView. The solution structure of the Nck2 SH3- 1 (2B86) was obtained from PDB and its NMR assignment with an accession code of 6 854 was downloaded from BioMagResBank. The graphic software