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Nascent Hairpins in Proteins: Identifying Turn Loci and Quantitating Turn Contributions to Hairpin Stability Jordan M Anderson, Brice Jurban, Kelly N. L. Huggins, Alexander A. Shcherbakov, Irene Shu, Brandon L. Kier, and Niels H. Andersen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00732 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 12, 2016

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Biochemistry

Nascent Hairpins in Proteins: Identifying Turn Loci and Quantitating Turn Contributions to Hairpin Stability §

§

Jordan M. Anderson , Brice Jurban , Kelly N. L. Huggins, Alexander A. Shcherbakov, Irene Shu, Brandon Kier, and Niels H. Andersen* Department of Chemistry, University of Washington, Seattle, WA 9815

Abstract Many factors influence the stability of hairpins that could appear as foldons in partially folded states of proteins; of these, the propensity of certain amino acid sequences to favor conformations that serve to align potential β strands for antiparallel association is likely the dominant feature. Quantitating turn propensities is viewed as the first step in developing an algorithm for locating nascent hairpins in protein sequences. Such nascent hairpins can serve either to accelerate protein folding or, if they represent structural elements that differ from the final folded state, as kinetic traps. We have measured these “turn propensities” for the two most common turn types using a series of model peptide hairpins with 4- and 6-residue loops connecting the associated β strands. Loops of 4 – 6 residues with specific turn sequences containing only natural L-AAs and glycine can provide as much 15 kJ/mol of hairpin stabilization versus loops lacking the defined turn loci. Single-site mutations within some of the optimal connecting loops can have ∆∆G effects as large as 9-10 kJ/mol on hairpin stability. In contrast to the near universal II’/I’ turns of model hairpins, a number of hairpin-supporting XZZG-sequence β-turns with αR and/or γR configurations at the ZZ unit were found. A series of turn replacements (4-residue β-turns replaced by sequences that favor 5- and 6-residue reversing loops) using identical strands in our model systems have confirmed that several sequences have intrinsic turn propensities that could favor β-strand association in a non-native strand register and thus serve as kinetic traps. These studies also indicate that aryl residues immediately flanking a turn sequence can alter relative turn propensities by as much as 9-11 kJ/mol and will need to be a part of any nascent hairpin recognition algorithm. §

Contributed equally to the experimental data acquisition and data analysis appearing in this article.

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Introduction The pathways involved in protein folding are the key to a more detailed understanding of protein folding landscapes and misfolding behavior. Amyloid-related misfolding occurs for both intrinsically unfolded peptides and from partially-unfolded states of proteins1–3. Folding pathways can frequently be viewed as the formation of relatively stable local structural units (“foldons”) which then coalesce by forming more native-like tertiary contacts by presumably random diffusion/collision interactions4–7. The candidates for foldons include short α helices, β turns, local hydrophobic clusters, and possibly short β-hairpins. The formation of such local foldons could even be pre-equilibrium events prior to entering the folding transition state ensemble. Indeed, there has been some success in protein fold prediction based on foldon stability analyses and sequential structuring8,9. Among these potential foldons, α helices are well understood but hairpins less so. Studies looking closely at isolated parts of proteins, particularly secondary structure elements that could represent a pathway element or be part of the transition state ensemble, have been quite prominent over the past two decades. Much is understood about helices and their stabilizing interactions10–14. Indeed, the guidelines for a priori design of helical peptides outside the protein context are well developed; there are algorithms and computer programs that are quite successful for predicting intrinsic helicity based on residue propagation/nucleation values as well as specific N-capping and C-capping interactions for each residue15–21 . Phi analysis, as developed by Fersht, has been the primary method for mapping protein folding transition state ensembles on an individual residue level, using free energy relationships22. Near unity mutational Phi values indicate structuring at the transition state; while values near zero imply that a particular residue has not achieved a native-protein-like environment at the folding transition state. In some cases, however, Φ values outside of the 0 – 1 range are observed. Such aberrant Φ values (< 0, > 1) can reflect a non-native structure in the transition state. The ability to predict the “intrinsic” helicity of sequences has provided a number of insights into the folding pathways of proteins (and potential kinetic traps), including the rationalization of anomalous Φ values. Indeed, Merlo et al.23 found that all ∆∆GF‡ values for mutations in a CI2 (chymotrypsin inhibitor 2) helix, including sites with aberrant Φ’s, could be rationalized based on calculated intrinsic helix propensities. Presumably this reflects a helix at the transition state that lacks the native, or has non-native, tertiary interactions. Given the slower dynamics of β-hairpins and their demonstrated importance as both nucleation sites and kinetic traps (vide infra), we expect that an algorithm that identifies sequences of proteins that will


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readily form hairpins could be extremely useful. This account is our first of a series aiming at deriving such an algorithm. Beta hairpins, the simplest model representations of β sheets24–26 , with folding rates on a µs time scale27–31 , are likely formed along the path to overall protein folding for many β and α/β proteins. Phi analysis, in conjunction with mutational studies32 has suggested that β hairpins can form early in the protein folding pathway and may serve as nucleating sites in the “diffusion/collision” processes33 where secondary structures form early followed by establishing tertiary contacts and then reaching the native fold, or a “nucleation condensation” mechanism34 with some secondary and tertiary structural elements forming in tandem with the collapse of the whole protein around an extended structural nucleus. Hairpin redesigns within β- and α/β-proteins have been shown to improve stability, particularly in the case of reversing loop mutations that increase turn propensity35,36 . In some cases such alterations have changed folding mechanisms36–39, but instances of both enhanced35– 37,40–42 and reduced protein folding rates37,41,43 have been reported for mutations that include stable hairpin inserts. An example from Prof. Baker38 , in which hairpin redesign altered the early-formed hairpin in a four-stranded sheet consisting of two hairpins is notable. Studies of ubiquitin provide examples of both folding acceleration and folding frustration (vide infra) by adjusting or inserting redesigned hairpins41,43 . The use of β hairpins to better understand these protein folding pathways has increased since the successful isolation of some monomeric, stable hairpins from β- and β/α-proteins44,45. While it is now established that these initial examples of protein hairpins with intrinsic hairpin formation tendencies are not as well-folded as initially reported29,31,45 , subsequent studies of designed hairpins have resulted in systems that have much higher fold stabilities. These improvements have included the use of improved turn sequences, NPATGK46,47, DPETGT48 , IpGK (p = D-Pro)49–52 , specific cross-strand interactions31,46,47,53 , and β-sheet capping interactions54,55. With these improvements a number of hairpin systems with fold populations ≥ 90 % (in water at ambient temperature) have been reported and some of these are used in the present study to examine the effects of specific turn region mutations on turn propensity and turn type selection. Designed hairpin models are now available for the three hairpin conformations commonly found in proteins (Figure 1). Henceforth we will be using the residue numbering system51,55 we introduced46 in 2004 and the turn classification system of Thornton57,58; both appear in Figure 1. For each class, isolated hairpin stability in aqueous medium is governed by a number of factors which would need to be incorporated in an algorithm for scanning protein sequences for likely sites of early hairpin formation: Coulombic effects due to the end charges (and, in some cases,


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cross-strand sidechain charges), the “β-propensities” of the S ± n (n > 1) residues, specific hydrophobic or aromatic cluster formation by cross-strand sidechains, and, finally, the “turnpropensity”. Citations for studies of these factors appear in our most recent discussion31 of hairpin stabilities and dynamics.

Figure 1. The three common hairpin types in proteins (nomenclature as described by Thornton57,59 which have also been prepared as stable hairpins outside of protein contexts; the residue labeling scheme employed herein is shown. The S residues are in “strands”; while the T-residues are the turn loci46 .

The S ± 1 residues are typically considered to be part of the turn. Thus in the case of [2:4]and [2:2]-hairpins (the latter have an additional H-bond between the S ± 1 residues), residues S−1/T1/T2/S+1 constitute a β-turn. The earliest efforts to excise, and improve, hairpins from protein contexts illustrate these three systems: the 2nd hairpin of the B domain protein G (GB1p) is a [4:6]-hairpin (with a DDATKT turn sequence) in both the protein and as the isolated peptide44,60, the N-terminal hairpin of ubiquitin is a [3:5]-hairpin which also forms to a limited extent for the isolated sequence61 , and tendamistat-(15-23) is a [2:4]-hairpin with a distorted SWRY type-I β-turn. In the case of tendamistat the excised native fragment failed to display βhairpin characteristics62 , but it was nonetheless the basis for a series of designed hairpins63–68 . In natural WW domains40,69 both six- and five-residue turns are found in the fold-nucleating 1st hairpin and comparably well-folded analogs with type I’ and II’ β-turns have also been prepared35 . Changing the turn type can have far reaching consequences on structure; turn effects go beyond just providing net fold stabilization and a possible folding nucleation site. In the case of [2:4]-hairpins, proper β-strand association is favored for type I’ (αL at T1, γL at T2) and II’ (ΤL at T1, γR at T2) turns, see Figure 2 for the locations of these conformations on a Ramachandran plot. In our discussion, we use only the [2:4] designation even for systems designated as [2:2]hairpins by Thornton and co-workers54-56, 66. A number of studies58,70–72 have noted different turn residue-probability statistics within hairpins versus turns in other loops. Such preferences appear be even greater in short peptide constructs than in proteins. Although turn preferences in protein hairpins had been defined in these earlier studies57,70,73 , this was not recognized in two early


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attempts to improve hairpin fold stability which, as a result, provided examples of switches in turn type. Blanco et al.63,64,66 employed an NPDG-turn, the most common type I (not I’) turn based on residue location statistics70, to connect the tendamistat strands. Later studies65,67 established that the NPDG unit in these hairpin designs was preferentially incorporated in a [3:5]-turn with the glycine at the T3 bulge site. Searle24 attempted a replacement of the [3:5]turn, TLTGK, in the isolated N-terminal hairpin of ubiquitin (·· K-TLTGK-T·· ) with an NPDG sequence. A more stable hairpin resulted, but there was an alternative β-strand association register with a well-formed NPDGT [3:5]-turn: a strand residue (T) moved into the reversing loop once again placing Gly in the bulge site. When the same replacement was carried out on the complete protein43 , the native hairpin register was retained but the fold stability was decreased by 9 kJ/mol and the (TLTGK) → (NPDG) mutation resulted in a 30-fold decrease in the folding rate. This stands as a clear case of folding frustration due to an intrinsic non-native hairpin preference that can appear prior to the folding transition state and increase the barrier to the transition state ensemble that leads to the native structure well. We expect that this could be a common result when there is an intrinsic preference for a non-native hairpin conformation and is another reason to develop an algorithm for identifying nascent hairpins in protein sequences. Four residue turns with an αR-γR T1-T2 conformation (a type I β-turn) are found in protein hairpins and the NPDG sequence is represented in this group. Figure 2 compares the T1-T2(S+1) phi/psi values of these hairpins with those at T1-T2-T3 of the closest analogous [3:5]hairpins.

Figure 2.

A Ramachandran plot illustrating the

phi/psi angles in two types of protein hairpin turns with a strong statistical preference for a PDG sequence within the turn. Both classes have type I turn preferences (αR - γR) at PD. In the [3:5]-Glybulge hairpins G is in a γL conformation. In hairpins with a four residue turn the Gly is in ε region of the Ramachandran plot. Three other regions, not shown on this plot appear in turns: αL in hairpins with a I’ turn, ΤL (φ = +60, ψ = −110°) in hairpins with a II’ turn, and the mirror image, ΤR, appears in type II βturns.

We anticipate that a commonality in sequence preference for a β-turn and [3:5]-turns could be the basis for folding frustration in many systems. Additional examples of NPDG insertions


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appear in the present study. Sequences with an S+1 glycine and much greater propensity to form a 4-residue turn have come from the present study (vide infra). The [4:6]-hairpins also have a common conformation over the six-residue turn span, -β-αR-αR-γR-αL(T4)-β- , of which the αL at T4 is the most restrictive, presenting yet another position with a strong preference for a Gly. We (and other groups) have found instances in which particularly significant hairpins in proteins have rather modest fold stabilities outside of the protein context24,29,36,61 . The native Pin1 WW domain provides a notable example: the 1st hairpin has a well-defined fold-nucleating role32,35,40,74 , but as the isolated peptide, the ∆GF300 value is unfavorable (on the order of + 4 kJ/mol)36 . Thus it appears that being able to recognize intrinsic hairpin folding propensities so modest as to support only 10 – 15% fold formation in the unfolded ensemble may still provide significant folding pathway insights. While an algorithm for searching protein sequences for the location of nascent hairpins (and scoring these for folding propensity) would need to include βstrand propensities and terms for cross-strand interactions, we view turn location and type forecasting as the first requirement and the best initial screen. Herein we report studies in which [2:4]-, [3:5]- and [4:6]-turn favoring units are placed within a set of common β-strands with hairpin formation quantitated by NMR. We also present mutational studies, at the turn loci of [2:4]- and [4:6]-hairpins, which provide ∆∆GU values for residues at each position. In the case of [2:4]-hairpins, we have included Aib75 and some D amino acid residues in the survey; these have previously been used to create particularly stable hairpin models49–54,76 .

Materials and Methods Peptide Hairpin Systems Examined Eight peptide systems were selected for this study (Table 1). With the exception of systems 1 and 2, all are shown with a 4-residue INGK or SNGK turn even though other variants were examined. Systems 3 – 6 have been examined previously53,54,56 and the folding diagnostics have been fully validated. Table 1 (and Table S1) lists the folding-induced chemical shift changes as 100% folded chemical shift deviations (CSDs in ppm) at the sites that accurately report the folded population. Using these values, the mole fraction of the folded state (χF) can be obtained by dividing the observed CSD by the 100%-CSD for each diagnostic site. The χF displayed in tables represents an average over the sites listed in table 1, with deviations representing the agreement of the individual probes. The resulting χF can be converted into ∆G values using the standard definition for reversible chemical equilibriums (∆GU = –RT(lnKU)), K = (1 – χF )/ χF . These values agree well with values derived from circular dichroism (CD) and HD exchange data, described below. A number of turn mutations were previously examined in these series and additional mutants were made for the present study. Systems 2 and 7 were prepared specifically for this study. In system 7, the cross-strand W/W pair of system 6 was moved out to the terminal non-H-bonded site and flanked by an attractive Coulombic pairing at the termini to create a βcap36,55,77 and provide additional β-strand sites for fold population analysis. Some mutated


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analogs of system 7 (KWLTVS-XXXX-KITVWE) proved to be not sufficiently soluble for ready NMR analysis; in those cases, a T13R mutation was introduced. In the three systems that were examined with and without the T13R mutation, the mutation had essentially no effect on the fold population. The effects of mutations within the loop of system 1 upon both fold stability and folding rates was reported recently31 . System 2 represents a longer strands version of system 1. System 8 is an extended version of the β-capped turn appearing in system 355 and displays comparable fold stability.


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Table 1. Hairpin systems used in this study with the 100%-fraction folded CSD values (in ppm), at 300K, shown for diagnostic sites employed for calculating the folded fraction (χF) value for analogs. The β-turn species are show with an NG turn locus; in each case, a more stabilizing locus such as pG ( D-Pro-Gly ) was also examined to more nearly reach a fullyfolded state. The sites of cross-strand H-bonded amides used as folding references are shown as bold residues; the downfield shifted Hα sites at non-bonded sites employed are shown bolded and underlined. Other residues that provide CSDs that can be used for fold quantitation are underlined. The SG naming refers to the Schenk-Gellman double hairpin78,79 .

Peptide systems S-3 HN

S+3 HN

S+5 HN

S-4 Hα

S-2 Hα

S+2 Hα

S+4 Hα

Hε3 / other / Cter-GHN

0.78

1.85

N/A

N/A

0.62

0.57

N/A

-2.43 (W3)

1.40

0.95

N/A

0.75

N/A

0.85

N/A

-2.4 (W13)

Ac-W-INGK-WTG-NH2

N/A

N/A

N/A

N/A

N/A

-0.19 N/A

N/A

4 KKLTVS-INGK-KITVSA

1.25

1.20

1.25

1.06

1.06

0.72

0.91

-2.0 (W6) -3.4 (G8) +1.3 (I2N) no Trp

5 KKLTVW-INGK-WITVSA 6 KKLWVS-INGK-KIWVSA 7 KWLTVS-INGK-KITVWE

1.65

2.06

1.43

1.45

N/A

N/A

1.26

-2.28 (W6)

1.49

1.24

1.38

0.79

1.07

0.74

0.58

N/A

1.10

1.01

1.45

1.08

0.85

0.63

1.11

8 Ac-WVS-INGK-KIWTG-NH2

1.52

1.49

N/A

N/A

1.01

0.77

N/A

Ac-VFIT-SNGKTYTE-VpGO-KILQ-NH2 SG1 Ac-VFIT-SNGKTYTE-VPGO-KILQ-NH2 SG2 Ac-VFIW-SNGKWYTE-VpGO-KILQ-NH2 SG4 Ac-VWIT-SNGKTYWE-VpGO-KILQ-NH2

1.04

1.14

N/A

0.58

1.13

0.90

N/A

0.94

0.97

N/A

0.62

1.13

0.95

N/A

1.61

1.70

N/A

1.00

N/A

0.86

N/A

1.69

1.43

N/A

0.90

1.31

1.04

N/A

-2.56 (W15) +1.48 (L3N) -2.1 (W10) -3.0 (G12) no Trp 0.84(S5N) no Trp 0.87(S5N) -1.8 (W4) 0.83(S5N) -2.2 (W11)

[4:6]-turns

1 KTW-NPATGK-WTE 2 RWTV-NPATGK-ITWE [2:4] and/or [2:2]-turns

3

SG

In the case of system 2, replacement of the NPATGK turn with the classic 4-residue turn (INGK) and with two [3:5]-turn units, NPDGT and SADGR36,69, were examined. The contribution of individual residues in the NPATGK [4:6]-loop of system 2 were ascertained by measuring changes in the extent of folding associated with single site mutations, in some cases these were performed in a DPATGK- or HPATGR-turn context. For most of the four residue β-


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turn systems (3 – 8), mutation were performed only at the two-residue turn locus (NG, above). The one exception to the latter generalization, was numerous attempts to replace an entire INGKturn with a variety of XXXG units (including the NPDG and TKQG sequences). Replacements of the β-turns of the L3Y-mutant of system 4 with [3:5] and [4:6]-turn units (NPDGT, DPATGR, and HPATGR) had been examined previously56 and that data has been reanalyzed and extended to the SADGR-turn to derive ∆∆GU values for each replacement for inclusion in this report. Other turn type replacements examined are listed here: a) replacing NPATGK with INGK, EPDGK and NPDGT in system 1, b) replacing SNGK in the first hairpin of the Schenk-Gellman78 double hairpin (Ac-VFIT-SNGK-TYTEV-) and its Trp-substituted analogs (Ac-VWIT-SNGK-TYWEV- and Ac-VFIW-SNGK-WYTEV-) with NPATGK31 and analogous 6- and 5-residue turns, and c) replacing INGK with NPATGK and NAAAKK in system 6. Turn replacements did not alter of pattern of chemical shift deviations observed for βstrand sites. There were some minor variations reflecting changes in turn type: I’ versus II’ versus I conformations within the four-residue turns. These are presented in the Results/Discussion section and detailed in the Supporting Material. With the exception of the attempts to insert NPDG as a β-turn, turn replacements did not alter of pattern of chemical shifts observed for β-strand sites. The diagnostic increases in hairpin fold stability associated with hexaflouroisopropanol (HFIP) addition to 8 vol-% of the medium was retained in systems 4 - 6 when the β-turn was replaced by [3:5]- and [4:6]-turn units. Figure S1 shows the retention of this pattern of downfield shifts for the turn replacements as well as the retention of HFIP-induced folding enhancement in system 5. Peptide Synthesis All peptides were synthesized on either an Applied Biosystem 496 MOS or Liberty Blue (CEM) synthesizer using standard Fmoc solid phase synthesis. Preloaded Fmoc-protected Wang and unloaded Rink amide resins were used for synthesis. Peptides were cleaved from resin (0.1 mmol) using a cocktail of trifluoroacetic acid : triisopropylsilane : water (38:1:1, 9.5:0.25:0.25 ml) for 1.5 hr. The resin was filtered and washed with DCM, the resulting filtrate was concentrated in vacuo, crashed out and washed with cold (-20 oC) diethyl ether, giving the crude peptide. purified using RP-HPLC (Varian ProStar 220 HPLC, Agilent 21.2 x 50mm C18 column, 10 ml/min, Eluent A : Water with 0.1% TFA, Eluent B : Acetonitrile with 0.085% TFA), using a gradient of 10 – 50 %B over 19 min. Peaks were visualized at 215 and 280 nm with verification by mass spectrometry (Bruker Esquire ion trap with ESI ionization). The concentrated fractions were then lyophilized resulting in the purified peptide. In addition, the amino acid sequences of all peptides were confirmed by NMR, with NMR providing confirmations of hairpin fold formation and hairpin register as noted in the next section. Structural Characterization of Peptides The appearance of an alternating set of downfield shifted Hα and HN signals for the β-strand residues56 was our primary indicator of hairpin formation and these shifts became the major basis


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for generating hairpin fold populations (vide infra). Cross-strand pairings of Trp residues were included in peptide systems 1 – 3 and 5 – 8 to stabilize the hairpin fold. W/W pairings that immediately flank a turn sequence (S ± 2)-sites (systems 1, 3, 5) as well as Trp-pairings at the most terminal non-H-bonded site in a hairpin (systems 2, 7, 8) result in an edge-to-face (EtF) aryl cluster31,36,47,53–55,80 . In these clusters, the Hε3 site of the edge-indole ring experience a 1.9 – 2.7 ppm upfield ring current shift, changes to which are a sensitive measure of the extent of hairpin formation. The edge-Trp sites are identified and the limiting shift observed (or extrapolated) for full hairpin structuring appear in Table 1. Cross-strand EtF W/W clusters also produce, in most contexts, a distinctive exciton couplet CD signal31,36,45–47,55,77,80 . The exciton couplet maximum occurs at ∼228 nm, and can be as large as +600,000 deg. cm2 dmol-1. Although the amplitude of the exciton couplet varies significantly, the CD melt always provides a useful measure of fold stability (as a Tm-value) once the temperature dependence, a circa 0.2 % decrease per °C,31,36 , of the 100%-folded [θ]228 value is taken into account. A 100%-folded [θ]228 versus temperature plot can be generated from the slope expectation using NMR measures of folding to adjust an observed [θ]228 value to its 100% value. The derivation of an unfolded baseline is illustrated in Figure 5 (vide infra), a representative exciton couplet melt. The reported Tm is the temperature at which the line for the fully folded and unfolded states is found, the Tm represents where the observed [θ]228 value is half way between the two baselines. The complete characterization of numerous examples of hairpin systems 131,47 , 3 – 653,54 , and 855 have already been published. The additional analogs prepared herein, did not deviated from expectations based on those studies, see Supporting Materials including access to an in house chemical shift data base including all peptides prepared specifically for this study. We have also reported56 the effects of mutations in the NG/pG turn loci of the Schenk-Gellman double hairpin, Ac-VFIT-SNGK-TYTE-VpGO-KILQ-NH2 (systems SG and SG1, Table 1) as well as a complete four state analysis79 of the folding equilibria. The normally less stable 1st hairpin56,79,81 was stabilized by cross-strand W/W pair insertions for some studies (systems SG2 and SG4). Chemical shift comparisons, as CSD histograms, appear in Figure S2, where they are compared to the system (SG) lacking the cross-strand W/W insertions. These also serve to illustrate the placement of an indole ring over the turn locus in WIXGKW units (X = N or D-Pro) producing diagnostic upfield ring current shifts. The key data in the characterization of a number of the analogs of system 2 are given here. The backbone Hα and HN CSDs for the series 2 parent system (RWTV-NPATGK-ITWE) as well as the CSD melts for the HNs appear in Figure 3. The NMR shift probes selected for χ F measurements are highlighted with an asterisk.


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*

* *

*

Figure 3. Chemical shift characterization of series 2 peptides illustrated by the parent system with an NPATGK turn sequence. In the top panel, asterisks show the sites (Hα at Trp2, Ile11 and the two largest HN’s shifts) used for the fold analysis. The HN CSD melts are shown in the lower panel.

The other CSD employed for defining the extent of folding of analogs is the upfield shift of Hε3 of W13. There is an excellent correlation between the backbone CSDs and the extent of shielding at W13Hε3 due to β-cap formation (Fig. 4). The formation of the β-cap for all species was also indicated by the appearance of the usual exciton couplet. The amplitude of the couplet also correlated reasonably well with chemical shift measures of the extent of folding and the melts showed lower melting points for the less stable species (Fig. 5). Fig. 4. The correlation between the upfield shift at W13Hε3 and the sum of the four diagnostic backbone CSDs over a range of NPATGK turn mutants and solution pH values.


Biochemistry

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Page 12 of 44

There is a slight displacement of the pH 8 line in Fig. 4, which possibly corresponds to a decrease in the folded reference value of Hε3 within the terminal β-cap unit at the higher pH. We have not been able to come up with a precise explanation of this pH effect. Figure 5. CD melts as [θ]228 versus temperature for a series 2 analogs spanning from very stable (DPATGK and NPATGK which have different 100% folded ) to a case with the folded state nearly absent (NPAATK) as well as a derived unfolded state baseline ( - - - - - ). The insert shows the traces for the APATGK turn species.

T (oC)

The unfolded CD baseline ( - - - - - ) shown in Figure 5 was derived from the extend of folding observed for the nearly unfolded NPAATK analog in NMR experiments. To a first approximation this CD baseline should also apply to other peptide systems (such as series 7 peptides). Hairpin system 7 also has a Coulombic β-cap inserted to increase fold stability. This system proved to be very stable indeed. This can be seen in the Hα CSD melt for one member of the series (Fig. 6). As an additional indication of nearly complete hairpin fold formation, there was very little increase ( ∆CSD = + 4.2 ± 2.6 %) in the diagnostic strand CSDs (which include L3N, V5N, I12N, V14N and W15Hε3) either upon an IGGK → IpGK mutation or on addition of HFIP to the medium. The same also applies to the upfield W15Hε3 CSD. These features are


Page 13 of 44

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Biochemistry

illustrated in Figure S3 with NH and Hα CSD histogram comparisons. The Trp-Hε3 CSD observed (-2.67 ppm, upon adjustment to 100%-folded and extrapolation to 280K) was the largest observed for a β-capped hairpin to date. A time course of NH exchange experiment for the IGGK turn species in D2O at pD = 4.58, indicated a protection factor of 36 ± 9 at 280K corresponding to χ F = 0.973 ± 0.006 (the mole fraction of the folded state). Details of this experiment (Figure S5, Table S2) and several additional figures of data for the series 7 hairpins appear in the Supporting Material.

Figure 6. The temperature dependence of the Hα CSDs of KW6-IGGK and Hε3 CSD of residue W15. In this case, as in others, the loss in CSD at the W15Hε3 ( − 11 % over a 40° change in temperature) is greater than that at strand Hα sites (− 4.7 % at the Thr sites). This reflects the great sensitivity of the ring current shift to minor vibrational changes in the indole/indole ring geometry. In this case it corresponds to what would be a 0.16%/°C loss in the 100% CSD for Hε3. The CSD loss at Hα sites represent a decrease in the fold population. Examples of less stable analogs, all with a I’ turn preference, displaying the same overall pattern of CSDs appear in Fig. S4.

Hairpin Fold Populations From β-strand residue CSDs -- The NMR probes include select backbone HN and Hα sites with large chemical shift deviations (CSDs) that reflect fold population for each system, the extrapolated 100%-folded (χ F = 1.0) reference values of which are shown in Table 1 (vide supra). The basis for these have already been presented53,56,79 for systems 1, 3-6, 8, SG and SG1. As noted above, the HN exchange protection factors measured for KWLTVS-IGGK-KITVWE provided the basis for extrapolation to 100% values in series 7. In the case hairpin series 2, the


Biochemistry

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Page 14 of 44

values were obtained from χ F-values estimated by fitting CSD melts and extrapolating to 260K to obtain the 100% folded CSD values. Based on NMR analysis of Cross-strand Trp/Trp Cluster Formation -- The edge-indole Hε3 proton shift has been shown31,53,55 to be a good diagnostic for hairpin fold population in the hairpin systems employed herein. The extension of this conclusion to hairpin system 2 appears in Figure 4 (vide supra). In systems that are fully-folded over the 280 – 310K span, we have observed a 1.5 – 2.8 % diminution in the Hε3 CSD with each 10 °C increase in temperature31,36,82; In the case of system 7, it could be measured (∼1.6 %, see Figure 6) and this is assumed to apply here as well for calculating χ F-values. Analyses based on Circular Dichroic Spectra As illustrated in Figure 5 (and Figs. S6 – S8), CD melts of the W/W exciton couplet appearing in peptide systems 1 - 3 and 5 - 8 provide Tm values. As noted, 100%-folded value of [θ]228 and the temperature dependence of this value have been established this provided χF values in two cases where the peptides were not sufficiently soluble for 2D NMR studies. The appearance of a β-CD signal (minimum at 215-218 nm, maximum at ∼198 nm) can also reflect fold population and has been established as a means for determining fraction folded 25,26,78,83–88 . This was applied to KKLWVS-ISNK-KIWVSA, a peptide with a tendency toward aggregation at NMR concentrations, for which the β signature overwhelmed the small exciton couplet in the CD spectra. For an accurate measure of fold population based solely on CD measured β-strand character, a fractional exciton couplet signal, a variably scaled CD of a truncated peptide control (Ac-AWSNGKWT-NH2), was subtracted from the CD spectra of the peptide affording just the β-strand signal which was then used to determine χF .

Results In order to get an initial indication of the relative effectiveness of β-turns versus [3:5]- and [4:6]turns in generating stable hairpins, we have examined turn replacement in a number of systems with strands of varying length and different stabilizing cross-strand interactions. In this study we adopted an INGK- (or SNGK-) turn as the reference for ∆∆GU calculations and included a stabilizing N → D-Pro mutation, analogs of an optimized [4:6]-turn (NPATGK), and a number of previously observed [3:5]-Gly bulge turn sequences (EPDGK, SADGR, NPDGT and NPDGK), as well instances in which a NPDG sequence would need to form a β-turn. We included analogs of the Schenk-Gellman double hairpin78 , two systems with a W/Wflanked turn (1 and SG2), as well as three systems (2, 7 and 8) with a designed β-cap including EtF W/W interactions. System 1 is a truncated trpzip system for which the NPATGK-turn sequence was the original optimized version47 . The Schenk-Gellman systems were previously optimized with an SpGK-turn56,78,79 ; systems 4 and 8 were previously optimized with an IpGKturn52,53,55,56,89 .


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Biochemistry

The extent of hairpin formation was determined by calculating χF based on the probes listed in Table 1. The error in χF from the different probes was typically on the order of 0.06 or less. The χF-values were converted to ∆GU values; the effects of some mutations and turn replacements are collected in Table 2 as ∆∆GU-values. The error in χF-values can translate to errors in ∆GU values as large as 1 kJ/mol. In all systems, the NG → pG mutations were hairpin stabilizing, typically by 3 – 4 kJ/mol. The [3:5]-turns were, with the exception of SADGR at pH ≥ 6.5, less hairpin-stabilizing than the reference INGK-turn systems. Turn replacements were also examined in the simplest capped turn we have prepared, AcW-turn-WTG-NH2 (system 3). With the INGK turn as the reference, we observed the expected fold enhancement with IpGK (∆∆GU = + 3.7 at 300K); the replacement of INGK with NPDG gave a peptide that had no detectable tendency to form hairpins (∆∆GU < − 6 kJ/mol at 280K). The NPATGK replacement was better tolerated, ∆∆GU ≅ − 3 kJ/mol at 280K. Efforts to insert NPDG as a β-turn in the systems in Table 2 were also uniformly unsuccessful. Table 2. Hairpin ∆∆GU effects of turn replacements at 300K (pH 6.5 unless otherwise specified).

System

2b

(L3Y)-4

6

SG/SG1

8

SG4

Turn IpGK SpGK

n.d.

+2.84

+3.4

+3.5

INGK SNGK

NPDG

≈ − 8.8

−5.4

NPDGT NPDGK EPDGK SADGR

−3.5b

−1.65

NPATGK DPATGR HPATGR NAAAGK NAAAKK

SG2

W-turn-W n.d. +3.7/+4.8

+1.4

+2.8

< −7

1

a

< −9 −2.8 −5.2 −0.2

−2.0

b,c

−0.7 −0.8 b −6.5 f −11 b

−2.2

−2.8

d

−4.3 −2.1 e

−4.0

−2.6

−2.3

+2.6

−0.4

< −9

−6.4 < −9

+3.0 +1.9

−2.6

-2.5 -4.3

a) Positive values indicate greater hairpin stability than that observed for the INGK/SNGK loop peptide sequence. b) At pH 2.5, where the solubility is greater, fold populations are in a more readily quantitated range. c) At higher pH the SADGR turn replacement appears to be as hairpin-stabilizing as the INGK and NPATGK loops. d) This ∆∆GU value for INGK → SADGR was measured in system 7. e) This ∆∆GU value for INGK


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Page 16 of 44

→ SADGR was measured in system 5. f) The HPATGR and HPATGK loops produce much greater hairpin fold populations when the His is fully deprotonated (vide infra); this condition does not necessarily apply at pH 6.5; the approximate pH for this observation. In the case of KKLWVS-NPDG-KIWVSA, the peptide was predominantly unfolded, but two hairpin states could be recognized and quantitated. Fig. S9 shows the CSD patterns that reflect [3:5]-hairpin formation. KKLWVS-NPDG-KIWVSA ↔ KKLWVSNPDGKIWVSA ↔ KKLWVS-NPDGK-IWVSA

[2:4]-hairpin (~9%)

coil (≥70%)

[3:5]-hairpin (20%)

Of these states, only the [2:4]-hairpin has the favorable cross-strand W/W placement that should provide hairpin fold stabilization; apparently this is not enough to offset the poor I’/II’-turn forming capability of the NPDG sequence. This is yet another example of an NPDG sequence preferring to include another residue in the reversing turn placing the glycine at the Gly-bulge position of a [3:5]-hairpin. We were, eventually, able to prepare hairpins with a 4-residue NPDG turn only with additional constraints favoring the corresponding register alignment. Peptide WVCKK-NPDG-TVCKWTGPK-NH2 (χ F ≈ 0.90 at 280K) serves as one example; in this case the disulfide served to freeze the register. There was, however, evidence of internal “fraying” near the turn region for this peptide. The other example, KWLTVS-NPDG-KITVWE (χ F = 0.24 at 300K), is from peptide series 7. Series 7 hairpins have a particularly effective β-capping unit, vide infra, that functions only with the correct register. The NPATGK [4:6]-turn and its analogs were less effective in favoring hairpin formation for most of the systems examined; this was particularly the case for the NAAAXX sequences. However for [4:6]-turns of this general type, the effects of a turn-flanking W/W unit is particularly large. The increase in hairpin fold stability is 2 – 4 kJ/mol in the case of the NPATGK loop; in the case of NAAAXX sequences, the difference associated with introducing the turn-flanking Trp’s increases to 6 – 9 kJ/mol. We had initially intended to use mutational studies of peptide HP7, system 1 when the turn is NPATGK, to derive residue-specific turn propensity values for [4:6]-hairpins. Already published studies31,47 include mutations to alanine at the N, P, T and G sites of the NPATGK turn: the ∆∆GU values observed at 300 K were: -6.4 (N→A), -0.8 (P→A), -1.2 (T→A), and –1.6 kJ/mol (G→A). However, the effects were not additive31, the NAAAGK mutant, which incorporates two substitution each of which was destabilizing as a single-site mutation (Σ∆∆GU = −2) was more stable than the NPATGK species (∆∆GU = +0.4). This, as well as the odd behavior of turn


Page 17 of 44

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Biochemistry

replacements in this and other W-turn-W systems (Table 2), prompted us to use system 2 for a more extensive study of both turn-site residue preferences in [4:6]-turns and for an examination of the effects of inserting alternative [3:5]- and [2:4]-hairpin forming turns. In system 2 (RWTV-NPATGK-ITWE), hairpin stability was enhanced by a β-cap rather than a turn flanking W/W pair. The parent peptide was quite well-folded (χ F = 0.88) at 280K with a hairpin population of χ F = 0.84 at 300K at both pH 6.5 and 8. At pH 2.5, the fold population at +

−

300K dropped to 0.69 reflecting the loss of the Coulombic component of the RW/WE β-cap. With only a few exceptions, each of which is discussed, a similar loss in fold stability was observed at pH 2.5 for turn mutants.

The ∆∆GU-values for loop replacements affording

alternative turn types appeared in Table 2; additional details appear in Table 3. Of all the hairpin systems examined, this was the system that displayed near equal fold stability for the best natural [2:4]-, [3:5]- and [4:6]-favoring loop sequences. A wide variety of both single site and double mutations (Table 3) were performed within the 6-residue loop and these served to define the residue-specific ∆∆GU values. Some of the less stable variants were not sufficiently soluble for NMR measures of the fold populations at all three pH values, but exciton couplet melts (as illustrated in Figure 5, vide supra) could be performed at pH 6.5 for all of the species prepared. As previously noted, a double mutant, with an NPAATK loop, which had χ F-values of 0.07 and 0.04 at 280 and 300 K provided, by a small extrapolation, an unfolded baseline for the CD melting studies: [θ]228 (U) = +45,000 – 800(T, °C)°. For calculating Tm values, we first subtracted [θ]228(U) from the observed values and then calculated the ∆[θ]228 -value at 300 K associated with structuring based on NMR measures of folding. Extrapolation to 280 K, provided a 100 % folding ∆[θ]228–value for each analog; these are somewhat variable, ranging from +540,000 to +370,000 °; this kind of variability also applies for system 7. The 1.6 % drop in this 100 %-folded [θ]228 value for each 10 ° rise in T observed for the best folded system 7 species (Fig. S8) was applied to similar systems; and for less stable systems, as a ∼ 2 % drop in their 100 %-folded [θ]228 values for each 10° rise in T. Turning to specific effects noted for hairpin system 2 and its [4:6]-loop mutants, the following appear quite general. In the case of loops that do not contain ionizable groups, the fold stability always increases on going from pH 2.5 to pH 6.5, with a small further increase on going to pH 8 in some, but not all, cases. This reflects the Coulombic component of the RW / WE

−

capping interaction that disappears upon glutamate sidechain protonation. Additional pH effects are introduced with ionizable sidechains in the loop region. It has long been known that Asx is 43,45

the optimal first loop site

. The fold stability increase from pH 2.5 to 6.5 to 8 increases with


Biochemistry

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Page 18 of 44

Asp versus Asn; this may reflect that complete deprotonation of all CO2H groups is not achieved at pH 6.5. Table 3. Hairpin fold populations and CD melting temperature for peptide 2 analogs having the RWTV-loop-ITWE sequence. (Deviations of the fraction folded values were found to be ± 0.08 or less, and represent the agreement of the probe CSDs.) Fraction Folded (χF) at 300K a

Loop Sequence

pH 2.5

pH 6.5

pH 8

Tm (oC) CD at pH 6.5

INGK

0.77

0.80

0.95

69.5

NPDGT SADGR NPDG

0.45 0.54 ≤ 0.08

n.s. 0.90 n.s.

n.s. 0.87 -----

72.0

0.69 0.64 0.04 0.46 0.38 0.20 0.18 (0.23) 0.29 0.13 0.55 0.03 0.49 0.32 0.73 0.55 0.45 0.04 0.05 ~0.05 0.18 ----0.55 0.66 0.42 0.63 0.42 0.40 < 0.15

0.84 0.81 [0.59] b n.d. 0.71 0.45 (0.56+) 0.47 (~0.3) 0.68 0.55 0.77 n.s. 0.88 0.66 0.92 0.88 0.83 0.35 0.15 ≤ 0.04 0.13 0.30 0.81 0.79 0.55 0.82 0.68 0.63 0.19

0.83 0.94 0.90 0.86 c 0.48 0.61 0.46 n.s. 0.49 0.35 0.83 n.s. n.s. 0.71 0.90 0.76 0.85 0.15 0.07 0.02 ----0.26 n.s. ----0.82 0.96 (0.96) 0.90 0.21

1 NPATGK 2 DPATGK 3 HPATGK 4 CPATGK 5 SPATGK 6 QPATGK 7 APATGK 8 GPATGK 9 IPATGK 10 TPATGK 1-2a NAATGK add T4A = NAAAGK 1-2v NVATGK 1-2g NGATGK 1-3v NPVTGK 1-4n NPANGK 1-4d NPADGK 1-4h NPAHGK 1-4a NPAAGK add G5T = NPAATK 1-4g NPAGGK 1-4v NPAVGK 1-6t NPATGT 1-6a NPATGA 1-6g NPATGG 2-6r DPATGR 2-3g DPGTGR 2-4s DPASGK add G5T = DPASTK


64.3 72.7 (> 80) [43-53] b n.d. 48.0 40.9 (44) 27.1 (37) 15.8 39.7 30.7 53.5 56.0 45.3 72.1 55.3 54.1 C0 ≥ S > I > Q > T > A > G > H . The best five (D through D0 are all well-folded −

representing only a 3 kJ/mol change in fold stability, while the D to A change has a ∆∆G greater than 7 kJ/mol. The S−1 position is clearly important for this hairpin class, but the exact ∆∆G varies, for example N → A: in this series is 4.4 kJ while it is 6.6 kJ/mol31 in series 1. The T3 position is the other site with a very wide ∆∆G range for mutations. With the +

exception of His , residues with polar sidechains were superior at this site with the previously selected Thr as the best one. The stability series determined for each position are collected in Table 4. Specific ∆∆G’s and data from which they were obtained appear in the Supporting Material (Table S3).


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Page 20 of 44

Table 4. Mutational effects on hairpin stability at positions in a six residue reversing loop.

Position

Ref. AA

S-1

N/D

T1 T2

∆∆G range

Stability Order

(kJ/mol)

-

D- > H0, C > N ≥ D0 > C0 ≥ S > I > Q > T > A > G > H+

>12

P

P≥AΕV>G

3.2

A

V>A>G

3.4 -

0

0

+

10

G

T > S ≥ N ≥ D > D > H ≥ V > A, G, H G > H0 ≥ H+ ≥ N > A ≥ T

K

KΕR≥T≥A>G

3.4

T3

T

T4 S+1

7

The sequence, NPATGK, which we derived46 from residue occurrence statistics remains, together with a 5-residue turn (SADGR) are particularly favorable choices for hairpin design outside of the classic β-turn type.

Only three clear instances of additional mutational −

stabilization were encountered: N → D /H0 and A → V. After the S-1 position, the next critical sites for hairpin stabilization are Thr at T3 and Gly at T4. The preferred Gly location, can be rationalized

based

on

the

established

φ/ ψ

preferences

for

[4:6]-hairpin

turns:

(β)−αR−αR−γR−αL−(β). The other relatively well-tolerated residues at this position, N and H, are, as it turns out, also tolerated at sites with positive φ-values in four-residue turns, vide infra. The rather stringent residue-preferences at S-1 and T3, with the exception of the I > A > G observation at S-1 (that corresponds to β-propensities), do not appear to be related to the established φ / ψ preferences at each site. It is established (see Supporting Material) that the φ / ψ preferences found in protein X-ray structures are also observed in these model peptides. The basis of these specific propensities appear in the discussion section: they reflect H-bonding interactions at the S-1 sidechain function. Next we turn to the more common [2:4]-hairpin turns. Although there is some prior data on turn propensity effects on hairpin stability for hairpins with type I’/II’ β-turns53–56,90 , much of it work from this laboratory, a more complete survey of ∆∆G effects of mutations within the turn locus was sought. We see two uses for this data, the design of stable hairpin models and incorporation into an algorithm for locating nascent hairpins in protein sequences. The most common means for insuring hairpin stability in designed constructs has been the inclusion of a Gly residue at T1 or T2. The Gly location, T1 versus T2, does, in many cases effect a change in turn type: II’( β-TL-γR-β ) to I’( β-αL-γL-β ). The trpzip species with an


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Biochemistry

ENGK45 versus EGNK90 serve as a classic example. The φ/ψ preferences at the T1 and T2 sites in these turns, TL = (φ = +60, ψ = −120°) 70 , are also the basis for including Aib75 and D-AAs in efforts to stabilize hairpins. D-Pro49,50,76, with a much narrower range of allowed φ/ψ values, favors both αL and TL conformations, the latter being a close match to a mirror-image poly-ProII configuration. There have been a number of hairpins that displayed a mixture of turn types in the folded state53 . Our primary emphasis here is to define sequences that favor turn formation consistent with hairpin formation rather quantitate propensities to form a specific type of turn (I’ versus II’, now extended to include some type I turns). Our presentation regarding [2:4]-hairpin turn variants, begins with extended series of mutations for systems that we have examined previously53,54,56 . The data for systems 3 and 5 will be presented first. Both of these contain W/W flanked turns. The prior studies were expanded to include additional substitutions within the NG turn locus. The changes in hairpin fold stability for system 3, Ac-W-IXXK-WTG-NH2 , are given below. ∆∆GF

χF

pn ≈ pP ≥ pa ≈ pG ≈ pA > UG > Nn ≥ NG > H+G >> PG (U = Aib) 0.8 2.3 1.1 0.3 1.1 ≥ 5.1 kJ/mol 0.90 0.868 0.72 0.62 0.58 0.47 0.09

Turns with D-Pro replacing the Asn residue at the T1 site resulted in a 2.6 to 3.4 kJ/mol stabilization of the hairpin state, with the heterochiral pP turn locus76 among the very best for hairpin stabilization53,54. Given the relative rarity of turns flanked by two aryl residues, and the previously noted anomalies associated with W/W flanking of turns31, we do not view the resulting ∆∆G values as particularly useful for nascent hairpin recognition, see below. A quite different trend was observed for system 5 (KKLTVW-INGK-WITVSA): with no hairpin stabilization noted for the inclusion of D-Pro at the turn locus. As it turns out, this reflects steric problems associated with the placement of the edge-indole ring in the specific edge-to-face indole/indole interaction that appears when there are turn-flanking Trp residues. These features and how they are distinguished from β-cap geometries, which applies for system 3, are the subject of another paper82 . NG > AG ≥ pG > GG > pa > pA > NK > pP >> AK >> NPDG System 5 bears some analogy to trpzip systems, in particular to the versions with WEGNKW45 and WENGKW90 turn sequences. The Kiederling laboratory has reported90 some turn site mutational effects for this system. The NG turn locus (type I’) appeared to be superior to GN (type II’). The total range of differences in hairpin ∆GU values from NG to pP was only 2.1 kJ/mol. However, the difference from AG to AK was 5.5 kJ (3.8 kJ/mol separate NK and AK) showing the importance of having at least one D-AA, G, or N within the turn locus. This provided our


Biochemistry

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Page 22 of 44

first measure of the extent to which these naturally occurring turn loci could favor hairpin formation versus representative sequences lacking an N or G residue at the turn locus. An attempt at replacing INGK with NPDG indicated a ∆∆GF value in excess of 8 kJ/mol The turn-flanking W’s are also quite important for hairpin stabilization82 . System 4, which lacks the tryptophan insertions, is considerably less stable: at 300K, the fraction folded for the INGK parent dropped from 0.74 to 0.30. System 4 (KKLTVS-INGK-KITVSA) was also examined53 as the slightly more stable (L3Y)-mutant and the distinctly less stable (T13A)mutant. As a result of the low intrinsic fold stability, we could not quantitate hairpin fold χFvalues for systems with turns less favorable than IANK. The trend in hairpin stability that resulted was, different than that system 3, notably by the placement of pX systems as distinct more folded than NG and AG. The stability ranking sequence bore greater similarity to system 6 (vide infra) which also lack a turn-flanking W/W unit. The ∆∆G effects of turn locus mutation for system 4 are: ∆∆G

pP > UG ≥ pG > pA >> NG > GG ≥ AG ≥ HG >> AN 0.5 0.3 2.5 1.2 2.2 kJ/mol

We also examined most of the system 4 analogs in aqueous HFIP (examples appear in Fig. S11), a medium that usually enhances hairpin folding. A 4.9 – 5.3 kJ/mol fold stabilization was observed upon HFIP addition with no change in the sequence of turn propensities. Some degree of hairpin stabilization could be effected by introducing a W/W pair at the S ± 4 positions, affording system 6 (KKLWVS-INGK-KIWVSA). At this cross-strand position the W/W pair does not adopt a single EtF aryl cluster geometry, and we expected turn propensity effects that would mirror those of hairpins lacking turn-flanking and other specific structureinducing cross-strand effects. The greater stability allowed us to examine and quantitate less effective sequences as turn loci; the results are summarized immediately below. For the four systems that were also examined in aqueous HFIP (8 or 20 vol-%), we observed a 3.4 – 4.1 kJ/mol stabilization upon HFIP addition, this is a bit smaller than that observed for the less stable system 4 hairpins (Fig. S11). ∆∆G

pG ≈ pN ≥ pA > AG > NG > GN > NN > AN > SN > AA ≈ NPDG 0.2 2.2 0.6 0.5 1.1 2.5 0.3 ≥ 2.6 kJ/mol

The only difference in stability trends (system 6 vs 4) was the case of AG versus NG. In system 6, the IAGK turn mutant was more stable by 0.6 kJ/mol while it was less stable by 1.4 kJ/mol in system 4. This suggests that there may still be some interactions between strand residues and the turn in system 6. Besides finding problems with the use of turn-flanking Trp residues, the take away from the studies above can be summarized as: 1) an NG turn locus is 2.6 – 3.4 kJ/mol less favorable than a pG turn, 2) that a number of turn loci with G or N at the T2 site are moderately to quite stabilizing – AG, GN, HG, NN, AN, and SN turns were evaluated, and 3) the absence of G or N at T1 and/or T2 was destabilizing by at least 2 kJ/mol.


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Biochemistry

A further increment in hairpin stabilization was required to evaluate less favorable turn sequences. That increment in fold stability could be gained by moving the W/W pair to the S ± 6 positions. This was combined with an (A16E)-mutation, to mimic the β-cap in system 2, affording system 7 (KWLTVS-turn-KITVWE). The fold population for system 2 with an IGGK turn was 0.964 at 300K (see Figs. 6, S5). Most of the systems examined were IXXK turn sequences. Some of the turn mutants were less soluble, particularly with replacements for the lysine in the turn sequence. In these cases, we examined the KWLTVS-turn-KIRVWE mutant. The T13R mutation, although it might be expected to be somewhat destabilizing due to the lower β propensity of R versus T, does not appear to effect any significant change in hairpin population in the systems examined with and without the mutation. The complete list of turn substitutions examined, and stability data for these mutants, appears in Table 5. Table 5. Hairpin populations of KW-LTVS-loop-KI-T/R-VWE peptide sequences (series 7). For 4-residue turns, the turn type (if not established as I’ or assumed to be that) is also shown. (Deviations of the fraction folded values, the variation in χ F over the multiple probes, were found to be ± ~0.06.) Turn type

NMR χF at 300Ka pH 2.5

pH 6.5

~0.98

> 0.97

0.92 0.91 0.93

> 0.96 0.964 0.945 0.94 0.93

~0.96 0.91 0.88

IHGK

0.89

~0.90

0.80

ITGK ITGK IGSK IGSK IGTK

0.51 II’ II’ II’

0.78 0.87 0.82

0.64 0.90 0.88 0.83

IGNK

II’

[ 0.85 ]

0.80 c

IGAK

II’

0.69

0.77

INNK

0.70

0.74

R13

INTK IAAK

0.57 0.13

Loop =

XNGX ANGK INGA YNGK

Loop =

IXXK

T13

IpGK

Mix

IpPK IGGK INGK IDGK IAGK

II’

R13 R13 R13

R13

0.72 0.91

pH 8

CD χF338K (pH) b

CD Tm

pH 6 (unless otherwise indicated) 0.75 0.80 (2.5) b 0.69 0.64 0.51 0.45 0.55 0.64 (2.8) b 0.10 (8.1) 0.07 0.21 ~0.41 n.d. 0.36 0.34 (6.5) 0.52 (2.5) b 0.31 (6.5) 0.34 (6.5) 0.36 (2.5) 0.05 (2.8) < 0.02 (2.5)

34 (2.8) GG > AG ≈ NG > DG > GS > GN ≈ NN This complete series represent a 5 kJ/mol change in fold stability at 338K. Based on prior hairpin systems this would translate to ∆∆G = 6.4 kJ/mol at 300 K. However, we expect that all turn effects on hairpin fold populations may be reduced in the presence of the very favorable βcap in the system 7 peptides. Both YRGR and YNGK were found to be among the most favorable hairpin forming turns. The YRGX and related turn loci were examined because of their occurrence in tachyplesin91 and gomesin92 antibacterial analogs that we were examining. Turning to the other end of the turn propensity spectrum, with the complete removal of Gly and Asn residues from the turn locus, for example the IAAK turn sequence, NMR diagnostics of hairpin formation remained (χF = 0.13 in water at 300K, including the upfield ring current shift of W15Hε3 for the terminal W/W cluster, and the CD exciton couplet. The CD exciton couplet observed had a significantly diminished


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Biochemistry

amplitude, and a much lower Tm was observed in the CD melt. This was also the case for four other radical turn replacements: GGGK, IGGG, INTK, and NPDG. In this series, HFIP addition did not increase hairpin formation appreciably and was thus not a means for extending the series to even poorer turn-forming sequences. Replacing the four-residue turn with optimized 5(SADGR) and 6-residue (NPATGK) loops, however, was well tolerated with only a 2.8 kJ/mol loss in fold stability versus the reference INGK turn. The IHGK turn sequence, which favors a I’ β-turn geometry is, even under typical biological conditions, about as favorable for hairpin formation than the best ‘natural’ II’ sequences, IGNK and IGSK. There is an ionization state effect on folding with the IHGK turn (∼ 3 kJ/mol more + favorable for H ), but this is smaller than those observed for hairpins with either an NPAHGK0 or HPATGK-turn sequence. For both positions within the 6-residue turns, H was superior for hairpin formation, by 1.6 and ≥ 11 kJ/mol respectively. The unfavorable effect of histidine deprotonation on the hairpin with the IHGK turn is greater at higher temperatures; this is clearly evident in the CD melt shown in Fig. S7B. In the IXXK series (with XX as the turn locus), the type-I’ XG systems are (with the exception of TG versus GT) superior to the type-II’ GX systems as hairpin stabilizing units. Indeed, IGTK, and even more so, IGSK are particularly good turn sequences and can be recommended for hairpin designs. Comparisons with IpGK, which is more stable, particularly at higher temperatures, are of questionable value since the pG locus can undergo an amide plane flip populating both type-I’ and -II’ turn conformations. Substitution effects at the S−1 and S+1 sites were also examined, but not as extensively. In the case of Type I’/II’ turns the S − 1 and S+1 sites have φ/ψ values close to β strand norms and the angles escaping from the turn locus are compatible with the twist of associated β-strands. Alanine is well tolerated at either position, but Thr, a classical high β-propensity residue is not favored, only tolerated only at the S − 1 position. The clearest example is the TGEK hairpin: the IGSK → TGEK mutation is destabilizing by 4 kJ/mol, but in the absence of a TGSK or TGNK reference we cannot determine whether the S − 1 Thr or the T2 Glu is the major contributor to hairpin destabilization for the TGEK species. The superior stabilizing effects of YNGK and YRGR turns may also, in part, reflect the greater β propensity of aryl amino acids. The exploration of mutational effects at the flanking S − 1/S+1 sites is not complete. We have, however, examined the other extreme, placing Gly at the S − 1/S+1 sites. Based on the -IGGGversus -GGGK- comparison Gly is better tolerated at S − 1. We did however find a number of quite favorable turn sequences that have a Gly at the S+1 position; these are presented in the next paragraph. As expected, throughout this series, additional glycine residues beside those required due to specific φ/ψ value requirements, are destabilizing particularly at higher temperatures. Another series (besides NPDG) of 4-residue loop sequence with Gly at the C-terminus was examined based on a recent report93 that the L38YVGSK-TKEG-VVHGVAT54 sequence within


Biochemistry

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α-synuclein forms a hairpin in a complex with a protein that inhibits it aggregation. The KTK(Q/E)G-V sequence appears 5 times as part of an 11-residue repeat in this amyloidogenic protein and may have a role in its amyloidogenesis94,95 . The PDB entry (4BXL) for the complex provides the following φ/ψ values for the TKEG turn region: -130/-154, -60/-51, -70/-35, +113/22°. The turn locus itself would be designated as αR, αR most closely related to a type I turn. We examined TKEG, TKQG, and TKKG (as well as a shuffled TGEK sequence that forms a type-II’ turn, vide supra and see the Supporting Material) as turns in the KWLTVS-turn-KI(T/R)VWE motif. The TKEG, TKQG and TKKG units support a hairpin conformation allowing β-cap formation quite well. The fold stabilities were reduced by only 1.8 – 4.2 kJ/mol relative to the INGK species and all were more stable than the TGEK turn species which forms a II’ turn from the same set of residues. The CSDs observed for β-strand sites of the TKXG hairpin species (see Fig. S10), however, suggested some distortions in the β-strand geometry near the turn region. These results prompted us to examine one of these new 4-residue loop sequences a replacement for NPATGK and other loops examined in the RWTV-loop-ITWE series (series 2, Table 3). The TKQG sequence, which appeared to be best folded in series 7 with at least 67 % formation of the β-cap interaction and a circa 0.8 hairpin fold population at 300 K, was nowhere near as effective for hairpin formation in series 2 in which the strands are two residues shorter. In series 2, the TKQG mutation was only 38% folded at 280K. Apparently the longer strands of system 7 allow the cap to form, even when the full length of the strands are not associated βstructures.

Discussion With the systems presented in this study the range of turn propensity effects on hairpin stability that have been examined is at least 12 kJ/mol with an additional 4 kJ/mol of stabilization available with the use of turn sequences incorporating non-protein amino acid residues. Even with the best β-caps that we have been able to design, there are certainly 4- to 6-residue sequences that will never be the reversing loop in a β-hairpin. It is likely that selected turn sequences in a protein structure could be providing as much as 16 kJ/mol of drive for nascent hairpin formation. However, actual hairpins in protein structures rarely employ the most favorable possible sequences for strand direction reversal. Since there are no cross-strand interactions of β-hairpins that provide this much enthalpic stabilization – the most notable ones are rather rare cross-strand aryl/aryl cluster formation which can be hairpin-favoring by as much as 6 kJ/mol, it is likely that turn location and propensity assessment will be a particularly good guide for fold and folding pathway prediction. Turns in fold-nucleating, β-hairpin structures in proteins, must rely on the intrinsic sequence β-propensities and turn propensity to form in an early transition state. In the final native fold, such β-structures can be stabilized by contacts with surrounding structural features. Previous


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Biochemistry

studies58,70 have relied on mining of the data within protein structure databases to determine site specific propensities. These may not be reliable representations of the propensities that apply to nascent hairpins, since many of the turns used may only be stable in the final structure after the folding transition: the hairpin stability is linked to folded-state contextual features. The β-hairpin systems presented here offer propensity data that is only determined by the turn sequences. Extensive mutational studies of designed hairpin models with high fold stability have allowed us to define the position-specific contribution of residues to effective hairpin formation for both [4:6]-loops and type I’/II’ β-turns and a more limited sampling of [3:5]-Gly-bulge forming units. We will be incorporating the turn position ∆∆G values determined in the present study into an algorithm for locating and stability ranking of nascent hairpins in protein sequences. In the [4:6] series, system 2, it was found that a number of mutations were well tolerated to the NPATGK turn, but the more stable [4:6]-hairpins had residues similar to the parent species in the turn region. This is likely due to the existence of specific H-bonds involving sidechain functions within the reversing loop. It was found that at position S-1, an H-bond acceptor is strongly preferred. In the case of S-1 Asp at pH ≥ 6.5, only an H-bond acceptor role is possible. Placing a His at this position created a pH switch, unfolded at low pH and well folded at high pH (χF= 0.04 and 0.9, pH 2.5 and 8 respectively). Turn site T3, also appears to prefer polar residues with Lewis basicity (potential H-bond acceptors) but the case is not as clear cut. Threonine remains as the best residue for this site, particularly based on melting point comparisons. Our

Figure 7: (A) A representative structure of System 1-NPATGK (PDB: 2evq)47. H-bonding shown with dashes, with position S-1 and T3 colored in green for clarity. (B) Example structure of the [4:6] turn found in hairpin 2 of the Nedd4-1 WW domain. (PDB: 5AHT, Turn seq. = DHNTKT)96.

observations concerning polar function preferences in the loops of [4:6]-hairpins prompted an


Biochemistry

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Page 28 of 44

examination of the well-defined structures with this feature, with an emphasis on obtaining a survey of H-bond occurrences. In 6-residue reversing loops, the backbone doesn’t remain flat but folds back on itself. This has the effect of pointing the HN protons of positions T2 to S+1 towards the inside of the turn (Figure 7) providing a number of possible H-bonding patterns within the loop; with polar sidechains, these too can be partners in the H-bonds. The turn-class defining HN of S-1 to the amide bond oxygen of S+1 H-bond is, sometimes, accompanied by a S+1 HN to S-1 O’ interaction. The S-1 O’ can also be quite close to the backbone HNs of T3 and T4. Of the three possibilities (T3, T4 and S+1), the T4 HN to S-1 O’ H-bond is a near universal observation. Figure 7A illustrates a bifurcated H-bonding situation with T3 and T4. Turning to sidechain function involvement in H-bonding networks. Panel A, shows a Hbond, S-1 Asn Hδ21 (the (E)-sidechain amide NH) to Thr-Oγ (T3 sc), that was a rare instance of the S-1 sidechain serving as an H-bond donor. Numerous possibilities with the S-1 sidechain as an H-bond acceptor appeared in the survey with the H-bond donor as one (or two) of the following: T2 HN, T3 HN and the OH of Thr at the T3 position. This could be the reason for T3’s preference for Thr, although the data suggests that an H-bond acceptor function at this sidechain site is important for stability. In any case, the proton donor capability of the T3 sidechain is not essential: NPADGK at pH 8 is as well folded as NPATGK (see Table 3). It is likely that the side chains can switch back and forth with minimal movement to the backbone structure. Our survey found one common instance of Thr-Oγ as a hydrogen bond acceptor; the donor is the S+1 sidechain when it is Thr Figure 8. Site specific residue preferences are or Asn. Specific residue preferences shown for the NPATGK [4:6] turn in series 2. found are illustrated in Figure 8, with ∆∆G data found in the supporting information (Table S3). In the case of system that can form [3:5]-hairpins, the studies were quite limited and specific mutational ∆∆G’s have not been derived. Future studies will likely focus of systematic mutations of a system with either an -SADGR- or -NPDGK- turn sequence. Mutations will be needed at each turn site. In the case of [2:4]- and [4:4]-hairpins, the present study has, in our view, provided sufficient data for initial parameterization of the turn-related portion of the nascent hairpin algorithm. Although the turn type, particularly type-I’ versus II’ is clear for many residue combinations at the turn loci, for the purposes of protein sequence searching, we expect that there will be only a single set of ∆∆GF increments. We will be treating all 4-residue “turns” as a single class rather


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Biochemistry

than deriving individual mutational effects for each turn type. As to glycine placement, the T2 position is clearly the most effective: compare IAGK to IGAK in series 7. The favorable hairpin formation for –XGSX- and, for example, -TKXG-, in series 7, was a surprising finding. Glycine placement at T1 is also favorable. But how much turn propensity increase should be attributed to glycine placement at the S+1 site? The TKXG-turn hairpins of series 7 are clearly a case where the β-capping interaction is the primary driving force for hairpin formation. Based on the inferior performance of the TKQG-turn in series 2 (and other comparisons), we expect that series 7 hairpins over-estimate the hairpin formation to be expected for less favorable turn sequences by about 5.6 kJ/mol. It is apparent that there are a number of complications that make it difficult to produce specific residue preferences for each site in the [2:4], [4:4] model. It seems that the placement of the glycine determines the preferences of the other sites. The complete folding of β-sheets is reliant on sequence-remote cross strand interactions, but these structures are, in many cases, nucleated by the folding of a single hairpin, followed by a collapse of the rest of the structure. Although there have been advances in computer programs to predict protein structures from sequences, we anticipate that the ability to identify nascent hairpins will result in solutions for significant problems in structure prediction. The turn propensities made available, by the present study, will serve as a necessary step in the design of an algorithm to locate nascent hairpins in protein sequences. The current study provides additional data suggesting the possibility of nascent hairpin formation, in which the favored hairpin has a different register, corresponding to [2:4]- versus [3:5]- versus [4:6]-turns, favored are likely to represent kinetic trap possibilities in protein folding. A nascent hairpin fold stability algorithm will be a tool for identifying systems for which this folding problem can be expected and tagging them for further study. Corresponding Author *Department of Chemistry, University of Washington, Seattle, WA 98105. E-mail: [email protected] Funding Source Information These studies were, over the years, supported primarily by the NIH (GM059658 and GM099889) but also by the NSF (CHE-0650318 and CHE-1152218) with all grants awarded to N.H.A.


Biochemistry

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Biochemistry

Xxx

For Table of Contents Use Only

Xxx

Xxx Xx x n Gly As

Ser

Thr

Gly

Pro

Lys

Ile

Asn

His

Glu

Site speciﬁc ∆∆Gs


95% Folded

58% Folded

Asp

Ly s

Ile

s

Thr

Gly

Turn Mutation

Glu

Ly

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*

* *

*

T (K)


Biochemistry

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o

T ( C)


Biochemistry

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Turn Position

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S-1

T4

A/G H+ / C o

T2

A/T

Intermediate

I/T/S/Q

G

Ho / N

Good

D / Ho / C-

V

Poor

β-Strand

N

P

A

T

G

K

A

N / S / D-

R+ / A

V

Do

Ro / T A/G

G

H/V A/G

T1

T3


S+1

β-Strand

Nascent Hairpins in Proteins: Identifying Turn Loci ... - ACS Publications

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