Charge-modulated accessibility of tyrosine residues for silk-elastin copolymers cross-linking

Abstract

The modulation of reaction kinetics with horseradish peroxidase (HRP)-catalyzed crosslinking of proteins remains a useful strategy to modulate hydrogel formation. Here, we demonstrate that the presence of positively charged lysines in silk elastin-like polymers (SELPs) impact the thermal transition temperature of these proteins, while the location in the primary sequence modulates the reactivity of the tyrosines. The positively charged lysine side chains decreased pi-pi interactions among the tyrosines and reduced the rate of formation and number of HRP-mediated dityrosine bonds, dependent on the proximity of the charged group to the tyrosine. The results suggest that the location of repulsive charges can be used to tailor the reaction kinetics for enzymatic crosslinking, providing further control of gelation rates for in situ gel formation, as well as the resulting protein-based gel characteristics.

INTRODUCTION

Control over the rate at which polymer crosslinks under physiological conditions to form hydrogels with different mechanical properties is important in many fields of research, including regenerative medicine¹, fibrosis, tumorogenesis², cell proliferation, drug and cell delivery, among other needs^3–4. There are many strategies utilized to crosslink protein polymers⁵, mainly focused on cysteine and lysine side chain reactivities⁶. These strategies can present challenges, as in natural and bioengineered protein designs these amino acids can also have alternative functions apart from crosslinking^7–8. Tyrosine plays numerous roles in protein functions, such as antibody recognition via the formation of pi-pi and cation-pi interactions⁹, in transduction cascades¹⁰, or for crosslinking to contribute to tissue stability (e.g., insect resilin, human aorta, bovine skin)^11–12.

In vitro, the enzymatic crosslinking of tyrosine (dityrosine formation) is a promising strategy for crosslinking natural and recombinant proteins leading to gel formation^{11, 13}. Horseradish peroxidase (HRP) is an Fe containing protein activated with H₂O₂ to form tyrosine free radicals that subsequently crosslink to form dityrosine bonds¹⁴. These reactions are relatively fast, take place in aqueous solvents under physiological pH and temperature with no toxic side products, which allows the incorporation of cells during the crosslinking reaction¹⁵ and the in vivo formation of cell-loaded hydrogels^16–18. Strategies to control of the reactivity and crosslinking kinetics with the tyrosines in these biopolymers remains to be more fully addressed. Some strategies have focused on the chemical modification of the enzyme itself by covalently attaching anthraquinone 2-carboxylic acid to the surface-exposed lysine residues to increase enzyme stability and activity¹⁹. Others have focused on controlling the reaction kinetics by changing ligand density¹⁵, pH²⁰ or enzyme/H₂O₂ concentrations²¹. These strategies, while valuable in reaction control in vitro, are difficult to apply in vivo. For example, pH is usually limited to near physiological values, in natural proteins ligand density is limited and invariable and in the body enzyme activity is difficult to modify in situ, although nonnatural enzymes can be used as an alternative^22–23.

Silk elastin-like polymers (SELPs) are genetically engineered proteins that combine thermoresponsive (GXGVP) pentapeptide domains found in the intrinsically disordered regions of tropoelastin^24–25 (where X stands for any amino acid except proline); and (GAGAGS) motifs found in the crystallizable (beta sheet) domains of the silkworm fibroin heavy chain protein from the silkworm Bombyx mori. Recombinant expression of these proteins offers control over molecular weight, composition, and allows the introduction of systematic modifications in structure. These features allow control of the protein chains and characteristics, thus, empowering SELPs as a valuable tool in fundamental studies of protein folding^26–28, for biomaterial scaffolding²⁹ and in drug or gene delivery³⁰, among other technological uses^31–32. However, the control of reactive moieties in SELPs towards crosslinking has not been extensively explored.

It seems reasonable thus to crosslink this material using HRP^{15, 26}, and exploit control over structure to tune both the crosslinking rates and the final mechanical properties of the hydrogels. The findings point out that the electrostatic complementary between enzymes and substrates are of crucial importance and determine the effectiveness of the reaction^{33, 34}. Based on the idea that complementary charges facilitate the enzymatic reaction by lowering the energy barrier³³, it is reasonable that charges of the same sign would hinder the enzymatic reaction and other possible interactions. Thus, this provides an understanding of repulsive electrostatic forces as a mechanism to increase the activation energy of the enzymatic reaction or interactions.

In this work, we present a systematic study of an efficient charge-mediated method to modulate three aspects of the assembly or properties of genetically engineered SELPs: (i) pi-pi interactions, (ii) HRP-mediated crosslinking kinetics and (iii) mechanics of the gels. This methodology, if applied in vivo, could also be useful to control reactions that take place with injected sol-gel biomaterials, such as for crosslinking or peptide cleavage in situ. Such control could be utilized to regulate the release of a bioactive peptide or modulate the durability or lifetime of the protein materials during tissue remodeling. This strategy could also be useful for modulating tyrosine interactions, such as between these bioengineered materials and cell receptors, to tailor binding kinetics to enable control of bioactivity³⁴.

MATERIAL AND METHODS

SELP PRODUCTION AND PURIFICATION

Gene synthesis, cloning and expression were carried out following our standard protocols³⁵. Briefly, SELPs genes were built through the assembly of oligos (synthetized by Genescript Biotech, Piscataway, New Jersey), subsequently cloned into a pET-29b(+) expression vector lacking the C’-terminal histidine tag and expressed in BLR(DE3) E. coli grown in autoinduction terrific broth (TB) medium (Grisp Research Solutions, Porto, Portugal) in a 2 liter bioreactor at 37°C (New Brunswick, BioFlo 3000) for 18 hours. SELPs were purified based on their thermoresponsive properties³⁵, with an average yield of around 350 mg of pure SELP per liter. Purity and amino acid composition were confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and proton nuclear magnetic resonance (1H-NMR).

DIFFERENTIAL SCANNING CALORIMETRY (DSC) AND TEMPERATURE MODULATED DIFFERENTIAL SCANNING CALORIMETRY (TMDSC)

Thermal analysis was performed with a TA Q-100 DSC (TA instruments, New Castle, Delaware, USA) under constant nitrogen flow (50 mL/min) using hermetically sealed Tzero aluminum pans and lids (TA instruments, New Castle, Delaware, USA). For each SELP, a volume of 25 μL (100 mg/mL in PBS, pH 7) was used, with a reference consisting of an equal volume of PBS. For DSC experiments, samples were heated from 15 to 60°C at 2°C/min. For TMDSC, a sinusoidal signal (0.32°C amplitude, 60 s period) was superimposed on the heating ramp. At least two independent experiments were performed for each SELP, and the values presented are the average of those measurements.

CROSSLINKING REACTIONS

For crosslinking reactions experiments, 50 mg/mL SELP solution in PBS (pH 7) was mixed with 10 U/μL of horseradish peroxidase (283 U/mg, SigmaAldrich, Ref: P8375–2KU). The reaction, performed at 37°C, was initiated by the addition of 1% v/v of a 0.75% v/v H₂O₂ solution.

FLUOROMETRIC ANALYSIS OF THE CROSSLINKING REACTION

Fluorometric experiments were carried out in a Synergy H1 Hybrid Multi-Mode Readerplate reader (BioTek, Agilent) using a 96 clear bottom black plate (Costar) at 37°C. The excitation wavelength was 315 nm; emission wavelength 405 nm for detection of di-tyrosine³⁶. As negative controls, SELPs without HRP and without HRP or H₂O₂ were used. Error bars represent the standard deviation of three independent measurements.

RHEOLOGY OF THE CROSSLINKING REACTION

Rheology measurements were carried out in an oscillatory rheometer (TA Instruments AR2000ex, New Castle, Delaware, USA) with SELP (50 mg/mL), HRP and H₂O₂ concentrations as above, using a 25 mm parallel plate geometry and a 1 mm gap. Prior to gelation, frequency and strain sweeps were performed to determine the linear viscoelastic region of the SELPs. Gelation experiments were carried out at 1 Hz frequency, 0.2% strain and 37°C. As a negative control the SELP without tyrosines (S2E8) was used (see Supporting Information).

RESULTS AND DISCUSSION

SELP PRODUCTION

Five different SELPs were produced (Table 1): one without tyrosines (S2E8) and four others (S2E8Y³⁵, S2E8Y-K1, S2E8Y-K3 and S2E8Y-K6) presenting a (GYGVP) pentapeptide motif regularly positioned in the SELP primary sequence. For those labelled with a K (-K1, -K3 and –K6), a positively charged lysine was placed before the first, third and sixth elastin-like pentapeptides in the sequences, respectively, thus a total of 8 lysines were present in these three variants, with none in S2E8 or S2E8Y. The SELPs were characterized by SDS-PAGE and ¹H-NMR prior to their use (see Supporting Information).

Table 1.

Name, amino acid composition, molecular weight and isoelectric point (calculated with ProtParam, www.expasy.org) of the SELPS used in this work.

SELP	AMINO ACID COMPOSITION	Mw (kDa)	pI
S2E8	MAMGGGA- [(GAGAGS)2(GVGVP)8]8-G	32.63	5.52
S2E8Y	MAMGGGA- [(GAGAGS)2(GVGVP)(GVGVP)(GVGVP)(GVGVP) (GYGVP)(GVGVP)3]8-G	33.14	5.52
S2E8Y-K1	MAMGGGA- [(GAGAGS)2K(GVGVP)(GVGVP)(GVGVP)(GVGVP) (GYGVP)(GVGVP)3]8-G	34.17	9.89
S2E8Y-K3	MAMGGGA- [(GAGAGS)2(GVGVP)(GVGVP)K(GVGVP)(GVGVP) (GYGVP)(GVGVP)3]8-G	34.17	9.89
S2E8Y-K6	MAMGGGA- [(GAGAGS)2(GVGVP)(GVGVP)(GVGVP)(GVGVP) (GYGVP)K(GVGVP)3]8-G	34.17	9.89

DIFFERENTIAL SCANNING CALORIMETRY (DSC)

DSC experiments (see Supporting Information) showed typical endotherms associated with the reversible phase transition of the SELPs²⁶, which arises from the loss of the clathrate-like (ordered) structures that hydrate the hydrophobic domains in the SELPs. The control SELP, S2E8 (i.e., lacking tyrosine and charged lysines), exhibited the highest T_t, above 41°C, in good agreement with the literature and the effect of the presence of silk domains on T_t^37–38. The SELP with tyrosines and without charge (S2E8Y) exhibited the lowest T_t and the highest enthalpy, corresponding to the presence of tyrosine, a hydrophobic guest amino acid³⁹. The SELPs bearing positive charges exhibit higher T_t’s and a lower enthalpy (Table 2). The presence of charged/hydrophilic moieties in the SELP backbone induced the displacement of T_t towards higher values, as the overall hydrophilicity of the SELP increased⁴⁰. The decrease in enthalpy was also related to the presence of these charged (lysine) moieties, as their hydrophilic nature reduces the number of water molecules that hydrate the SELP backbone⁴¹.

Table 2.

Transition temperatures and enthalpy values for the different transitions observed with the SELPs using DSC and TMDSC.

SELP	DSC		TMDSC
			TOTAL	REV. HEAT FLOW		NON-REV HEAT FLOW
	Tt (°C)	ΔH	Tt (°C)	Tt (°C)	ΔH	Tt (°C)	ΔH
S2E8	41.1	1.3	41.1	41.2	1.5	41.0	−0.2
S2E8Y	30.2	1.2	30.2	30.4	0.8	29.7	0.5
S2E8Y-K1	35.2	0.9	34.2	34.4	0.7	33.8	0.2
S2E8Y-K3	35.3	0.8	34.2	34.5	0.7	33.8	0.1
S2E8Y-K6	36.6	0.7	35.4	35.2	0.6	35.4	0.1

TEMPERATURE MODULATED DIFFERENTIAL SCANNING CALORIMETRY (TMDSC)

The endotherms with conventional DSC were the result of the sum of the different complex thermal processes, each following different kinetics. TMDSC experiments permitted the decomposition of the total heat flow observed by conventional DSC as the sum of non-reversible and reversible heat flows⁴², each of these associated with two different phenomena occurring simultaneously during SELP temperature-induced phase transitions. Non-reversible heat flow is related to the destruction of water clathrates that hydrate the SELP backbone, while reversible heat flow is related to the phase transition of the SELP backbone itself^43–44. For the tyrosine-lacking control SELP (S2E8), the non-reversible heat flow exhibited an endotherm peak, as a result of the destruction of ordered water structures that hydrate the SELP back bone; and an exothermic reversible heat flow, as a result of the reversible folding of the SELP⁴⁴.

For the SELPs bearing tyrosines, both non-reversible and reversible heat flow exhibited an endotherm peak. As previously reported⁴³, for SELPs a reversible heat flow endotherm is related to the destruction of ordered structures as result of the temperature increase. By comparison, among the tyrosine-containing SELPs and the control, the presence of the tyrosines capable of pi-stacking (i.e., forming ordered structures) were associated with the endotherm peak observed in the reversible heat flow^45–46.

The reversible heat flows of the different SELPs bearing tyrosines, as well as the effect from the location of the positively charged residue within the primary sequence of the SELPs was evaluated. When compared to the control, the presence of charged residues reduced the enthalpy associated with the reversible heat flow, with the effect more noticeable the closer the charged residue was to the tyrosine (Table 2). This result suggests that the closer the electrostatic repulsions are to the tyrosine the greater the reduction in pi-stacking of the tyrosines (thus weaker the pi-pi interactions).

FLUOROMETRIC ANALYSIS OF THE CROSSLINKING REACTION

We next studied HRP-mediated crosslinking of the SELPs (Figure 1). The onset of the sharp increase in fluorescence signal related to tyrosine crosslinking occurred at different time points depending on the SELP sequence, starting at shorter times for the S2E8Y (1 minute), with longer time frames for the SELPs bearing positive charges (3.25, 2.75 and 4.50 minutes for the S2E8Y-K1, -K3 and –K6, respectively). Interestingly, the maximum fluorescence found for the different SELPs was dependent on the presence and location of the positive charges. The S2E8Y fluorescence reached 5*10⁵ while for the S2E8Y-K1 and S2E8Y-K3, bearing positive charges distant to the tyrosine, the fluorescence values reached 3.5*10⁶ and 2.3*10⁶, respectively. For S2E8Y-K6, the fluorescence dropped to 1.5*10⁶. These results suggest the active influence of the repulsive positive charges on the HRP mediated crosslinking of the SELPs.

Figure 1.

Schematic representation of the effect of positive charges in the HRP-mediated crosslinking of the four SELPs used in this study: (A) S2E8Y, with a T_t far below 37°C, completely phase-transitioned and formed hydrophobic clusters that prevent HRP to access to crosslink tyrosines. For S2E8Y-K1 (B) and S2E8Y-K3 (C) the presence of positively charged lysines raises the T_t of these SELPs to 35°C, preventing full phase transitions. With time, the electrostatic repulsion between lysines and the molecular flexibility of the SELPs further exposes tyrosines for crosslinking. For (D) S2E8Y-K6, the repulsion between positively charged residues and their proximity to tyrosines prevents HRP-mediated crosslinks.

The early onset and the lower fluorescence values for S2E8Y are explained by the absence of positively charged motifs. The lack of positive charges in this SELP prevents electrostatic repulsion among molecules that otherwise hinder the formation of dityrosine bonds. However, the absence of lysines lowers the T_t of the S2E8Y (30.16°C) when compared to the rest of SELPs. This lower T_t supports the complete phase transition at 37°C for this SELP, leaving most of the tyrosines within the hydrophobic clusters formed by the phase transitioned elastin motifs and thus, inaccessible to the HRP.

The distance between the positively charged lysines and tyrosines has a significant impact over the crosslinking reaction. Charges located the farthest from the tyrosine (as in S2E8Y-K1 and –K3) exert the smallest effect over the initiation of crosslinking, while the effect was more prominent the closer the charge was to the tyrosine. Charges located adjacent to the tyrosine (as in S2E8Y-K6) delayed the onset of the reactions the most. When positive charges were located further to the tyrosines (S2E8Y-K1 and -K3), despite the delay in the onset of fluorescence, greater fluorescence values were attained. Interestingly, fluorescence values for S2E8Y-K1 and –K3 followed a similar two-phase increase, explained as result of the combination of three factors: increased T_t, repulsive effects of the positive charges and the high molecular flexibility (associated with the intrinsically disordered origin of SELPs). The increased T_t (≈35°C) for these SELPs means that they are not fully phase transitioned at 37°C, which would facilitate accessibility of the tyrosines to the HRP reaction. Thus, the first increase in fluorescence would result from the crosslinking of these accessible tyrosines. The second sharp increase (at 18 and 10 minutes, for S2E8Y-K1 and –K3, respectively) would be a combination of the repulsion between charges of the same sign and the intrinsic flexibility of the SELPs⁴⁷. Charge repulsion would be responsible of the molecular rearrangements that further expose tyrosines for HRP crosslinking, which could only be accommodated due to the intrinsic flexibility of the SELP backbone. If charges were located adjacent to the tyrosine, as in S2E8Y-K6, they exert an inhibitory effect over the HRP crosslinking reaction, when compared to the other lysine bearing SELPs. Repulsion between charges maintains the segregation of the tyrosines, hindering crosslinking, slowing the reaction rate, and limiting the number of crosslinks.

Fluorometric measurements of tyrosine crosslinking demonstrated that the presence of positively charged residues had a direct effect over crosslinking rate and the number of crosslinks formed, and this effect was modulated by the distance between the positive charges and the tyrosines. Charges far away from tyrosines had a small effect, while if adjacent, they were inhibitory. Intermediate location of the charges slows the reaction rate but permitted the molecular rearrangements that led to a higher degree of crosslinking.

RHEOLOGY OF CROSSLINKING REACTIONS

The HRP-mediated gelation of SELPs can be seen in Figure 2. For S2E8Y, the increase in mechanical properties was the slowest and the mechanical properties at the end of the experiment were the weakest of the SELPs studied (Table 3). For the SELPs bearing lysines, the increase in mechanical properties was faster and more significant than for S2E8Y. For S2E8Y-K1, the elastic modulus (G’) surpassed the loss modulus (G”) in the initial minutes of the experiment and subsequently, after 21 minutes, sharply increased to reach a plateau. For S2E8Y-K3 we observed similar behavior, the second sharp increase in mechanical properties was delayed when compared to S2E8-K1 (34 minutes). For S2E8-K6, mechanical properties increased with time following a logarithmic-like trend. Interestingly, for all four SELPs, the evolution of mechanical properties, as measured by rheometry and fluorometric signals related to the tyrosine crosslinks, followed similar trends. The lowest fluorescence corresponded with the lowest mechanical properties, as seen with S2E8Y, whereas the highest fluorescence values were obtained with S2E8Y-K1 and -K3, which corresponded to the highest mechanical properties. There is a correspondence in the trends between the fluorometric and the rheological values. For example, both S2E8Y-K1 and -K3 exhibited a 2-step increase in fluorescence, similar to that seen in rheological measurements. Moreover, the sharp increase in fluorescence and mechanical properties occurred at similar time points. This was also the case for S2E8Y-K6, which exhibited a similar logarithmic-like trend for the fluorometric and rheological values. These results corroborate our hypothesis on the effect of the positive charges over HRP-mediated gelation of SELPs.

Figure 2.

(Left) Fluorometric measurements of HRP-mediated tyrosine crosslinking for the 4 SELPs and their controls, SELPs without HRP and without HRP or H₂O₂, were used (purple and pink line, respectively). Error bars represent the standard deviation of three independent measurements. (Right) Rheological measurements of the HRP-mediated gelation of the (A) S2E8Y, (B) S2E8Y-K1, (C) S2E8Y-K3 and (D) S2E8Y-K6. In black, the elastic modulus (G’) and in red the loss modulus (G”).

Table 3.

Mechanical properties and fluorescence values at the end of the experiment for the 4 SELPs studied.

SELP	G’ (Pa)	G” (Pa)	Fluorescence (a.u.)
S2E8Y	86.7	5.2	0.5*106
S2E8Y-K1	47124.3	2922.1	3.5*106
S2E8Y-K3	7080.8	361.6	2.3*106
S2E8Y-K6	970.9	19.5	1.5*106

CONCLUSIONS

Here we demonstrated that the presence of charged residues within SELPs had a direct effect over the transition temperature and the enthalpy of phase transition. Moreover, the deconvolution of the total heat flow of the phase transition into reversible and non-reversible heat flows by TMDSC allowed us to identify the effect of the positively charged lysines over the pi-stacking capacity of the tyrosines present in the SELPs. This effect was inhibitory and more prominent the closer the charges were to the tyrosine. In addition, the presence of the lysines also had an effect on the HRP-mediated tyrosine crosslinking reaction rate, slowing it in the initial stages, but improving the overall performance of the reaction in all cases when compared to the control SELP. The increased transition temperature of the SELP variants bearing a lysine, in combination with the intrinsically flexible character of the SELPs, further exposed the tyrosines, resulting in higher degrees of crosslinking. The increased number of HRP-mediated crosslinks was dependent on the location of the lysine with respect to the tyrosine. These results suggest that the precise location of charges could be used as strategy (in vitro or in vivo) to control the biological accessibility of tyrosines in these types of protein polymers. Moreover, this regulatory effect could be extended to other enzymatic reactions, where repulsion among charges close to the reactive species or between the enzyme and the biomaterial could be used to tailor the speed at which the reaction takes place, leading, for example, to controlled in vivo hydrogel formation (or peptide cleavage) by enzymes in the tissue.