Báo cáo khoa học: Salt-induced formation of the A-state of ferricytochrome c – effect of the anion charge on protein structure

Salt-induced formation of the A-state of ferricytochrome c – effect of the anion charge on protein structure

Tác giả: Federica Sinibaldi, Maria C. Piro, Massimo Coletta and Roberto Santucci

Lĩnh vực: Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma ‘Tor Vergata’, Italy

Nội dung tài liệu: Nghiên cứu này khám phá sự hình thành trạng thái A của ferricytochrome c, một dạng trạng thái gấp khúc trung gian, dưới tác động của các anion có điện tích kép. Các anion này được cho là có khả năng liên kết với protein tại các vị trí bề mặt cụ thể, khác với hiệu ứng “bầu khí quyển ion” của các anion đơn hóa trị. Dữ liệu cho thấy các anion hai hóa trị có xu hướng ổn định trạng thái phối trí M-Fe(III)-H giống như trạng thái tự nhiên của protein hơn so với các anion đơn hóa trị. Nghiên cứu cũng xem xét liệu các anion hai hóa trị có thể liên kết tại các vị trí tương tự đã xác định trước đó cho các anion đa hóa trị hay không, bằng cách khảo sát hành vi của các đột biến K88E, K88E/T89K và K13N. Kết quả chỉ ra rằng các gốc đột biến này, góp phần tạo nên các vị trí liên kết anion đa hóa trị, đóng vai trò quan trọng trong việc ổn định cấu trúc tự nhiên của protein. Các đột biến này làm tăng lượng trạng thái H-Fe(III)-H bị sai lệch và khiến protein ít nhạy cảm hơn với sự hiện diện của anion so với cytochrome c loại hoang dã. Hơn nữa, các gốc này điều chỉnh cấu trúc của cytochrome c bị biến tính, ảnh hưởng đến trạng thái spin và sự phối trí với nhóm prosthetic.

Mục lục chi tiết:

• Structural information on partially folded forms is important for a deeper understanding of the folding mechanism(s) and the factors affecting protein stabilization.
• The non-native compact state of equine cytochrome c stabilized by salts in an acidic environment (pH 2.0-2.2), called the A-state, is considered a suitable model for the molten globule of cytochrome c, as it possesses a native-like a-helix conformation but a fluctuating tertiary structure.
• In this article, we extend our knowledge on anion-induced protein stabilization by determining the effect of anions carrying a double negative charge; unlike monovalent anions (which are thought to exert an ‘ionic atmosphere’ effect on the macromolecule), divalent anions are thought to bind to the protein at specific surface sites.
• Our data indicate that divalent anions, in comparison to monovalent ions, have a greater tendency to stabilize the native-like M-Fe(III)-H coordinated state of the protein.
• The possibility that divalent anions may bind to the protein at the same sites previously identified for polyvalent anions was evaluated.
• To investigate this issue, the behavior of the K88E, K88E/T89K and K13N mutants was investigated.
• The data obtained indicate that the mutated residues, which contribute to form the binding sites of polyanions, are important for stabilization of the native conformation; the mutants investigated, in fact, all show an increased amount of the misligated H-Fe(III)-H state and, with respect to wild-type cytochrome c, appear to be less sensitive to the presence of the anion.
• These residues also modulate the conformation of unfolded cytochrome c, influencing its spin state and the coordination to the prosthetic group.
• Formation of the unique, native structure of a protein occurs through well-defined folding pathways involving a limited number of intermediate species.
• In recent years, a large body of kinetic and equilibrium studies has provided extensive information on the folding pathway of proteins and led to the characterization of intermediate states, thus contributing to our understanding of the protein-folding mechanism [1-9].
• The non-native compact state of equine cytochrome c stabilized by salts in an acidic environment (pH 2.0-2.2), called the A-state, is thought to be a suitable model for the molten globule of cytochrome c; it possesses a native-like a-helix conformation but a fluctuating tertiary structure [10-14].
• With respect to the native protein, in the A-state some interior hydrophobic residues become exposed to the solvent [15], the W59-one-heme-propionate hydrogen bond is impaired (although the tryptophan remains within a hydrophobic environment) [14], and the heme-polypeptide chain interaction is reduced.
• Also, the hydrophobic core (which is composed of the two major helices and the heme group) is preserved in the A-state,
• Abbreviation CT, charge transfer.
• Anion-modulated structure of cyt c A-state
• stabilized by nonbonded interactions [12,16], whereas the loop regions appear to be fluctuating and partly disordered [12].
• The A-state is promptly achieved at pH around 2.2 upon addition of a salt to an aqueous HCl solution containing denatured cytochrome c; this has been ascribed to a screening action of the anions, which stabilize the compact form by binding to the positively charged groups on the protein surface [11].
• Recently, we investigated the role played by monovalent anions in promoting the transition from the acid-denatured protein to the A-state [17,18].
• Our results showed that the salt-induced A-state of ferricytochrome c is characterized by a variety of high-spin and low-spin states (where ‘high’ and ‘low’ stand for the S = 5/2 and S = 1/2 spin states of the heme iron, respectively) in equilibrium; in particular (at least), two distinct low-spin species, differing in their axial ligation to the metal, coexist in solution: a form with the native M-Fe(III)-H coordination, and a bis-histidine coordinated species.
• The equilibrium between these two low-spin forms, here indicated as M-Fe(III)-H H-Fe(III)-H is strongly influenced by the type of anion in solution [17,18].
• Because structural information on partially folded forms is important for a deeper understanding of the folding mechanism(s) and the factors affecting protein stabilization, in this article we extend our knowledge on anion-protein interactions by determining the effect on the protein produced by anions carrying a double negative charge.
• This is an interesting point to investigate, because, unlike monovalent anions (which are thought to exert an ‘ionic atmosphere’ effect on the macromolecule), divalent anions (as well as polyvalent groups, such as polyphosphates [19,20]) are supposed to bind to the protein at specific surface sites [21,22].
• Results Horse ferricytochrome c
• CD measurements
• Far-UV CD (200-250 nm) is a probe for the formation of the A-state from acid-denatured cytochrome c, as the A-state possesses a native-like a-helix structure [11,17].
• Figure 1 shows the gradual recovery of the ordered secondary structure in acid-denatured ferricytochrome c upon addition of increasing amounts of sulfate and selenate; the divalent anions stabilize the A-state at significantly lower concentrations than those needed for stabilization by monovalent ions [17].
• As shown in Fig. 2, the A-state tertiary conformation is less packed than that of the native form; the protein displays a weaker near-UV CD spectrum (Fig. 2A), and a weaker Soret CD spectrum (Fig. 2B).
• In this last case, the decreased intensity of the 416 nm Cotton effect is indicative of a perturbed heme pocket region, as the 416 nm dichroic band is considered to be diagnostic for the Met80-Fe(III) coordination in native cytochrome c [23,24]).
• As the M-Fe(III)-H coordinated species alone contributes to the dichroic signal, a significant population of macromolecules is expected to lack M80 coordination to Fe (III) in the A-state (it must be noted, however, that the signal is stronger than that recorded in the presence of monovalent anions [17,18]).
• The intensity of the 416 nm dichroic band is ~35% that of the native state, consistent with heterogeneity of the A-state.
• On the basis of earlier data (relative to monovalent anions) [18], a mixture between Met80-Fe(III)-His18 coordinated species and X-Fe-His18 miscoordinated species (where X represents the endogeneous ligand coordinated to the metal in place of Met80) is expected in solution.
• Under the conditions investigated, a histidine (His26 or His33) is expected to be the best candidate for ligand X (the other likely candidates, i.e. the lysines, are fully protonated at pH 2.2) [18].
• The heterogeneous character of the A-state prompted us to investigate the effect of sulfate and selenate on the heme pocket conformation.
• As shown in Fig. 3, the 416 nm dichroic band gradually increases (towards negative ellipticity values) with anion concentration, up to 4 mm anion; it then remains unchanged (up to 40 mm anion).
• This behavior markedly differs from that displayed by the protein in the presence of monovalent anions (the effect of perchlorate is illustrated in Fig. 3 for comparative purposes).
• The changes in the Cotton effect strength observed at high monovalent anion concentrations have been attributed to a shift of the M-Fe(III)-H ↔ H-Fe(III)-H equilibrium towards formation of the bis-H species [18].
• Thus, the data in Fig. 2 indicate that divalent anions have a stronger tendency to stabilize the (native-like) M-Fe(III)-H coordinated form.
• Unfolded macromolecules and peptides attain a degree of structure at temperatures lower than room temperature.
• We have recently shown that the A-state induced by monovalent anions displays a temperature-dependent 416 nm Cotton effect (temperature range: 25 °C to 2 °C) [17].
• In the present study, the investigation, extended to divalent anions, confirms that the native M-Fe(III)-H bond (indicative of a more structured conformation) is stabilized by low temperature (data not shown), indicating that protein flexibility hinders methionine coordination to the heme iron [25].
• Electronic absorption
• The 695 nm absorption band is considered to be diagnostic for the M80-Fe(III) axial bond in native cytochrome c [26].
• Figure 4 shows the effect of sulfate and selenate on acid-denatured cytochrome c, investigated by following the changes in the 695 nm absorbance band.
• It appears clear that both anions favor protein collapse into a compact form, and induce formation of a consistent population of macromolecules (~35% in sulfate, ~28% in selenate) with native M-Fe(III)-H coordination.
• These data are in excellent agreement with CD measurements and provide independent evidence for heterogeneity of the A-state.
• A-state stability
• Figure 5 shows the thermal denaturation profiles of the A-state of cytochrome c, as obtained from ellipticity values at 222 nm.
• As previously observed for monovalent anions [18], the shape of the unfolding profiles features a multiple state transition, as (at least) three distinct thermodynamic states are detected.
• The profiles clearly show that protein stability strongly depends on anion concentration; this highlights the primary role played by the anion-protein interactions in A-state stabilization.
• Competition among anions
• To better define the effect produced by monovalent anions on the sulfate-induced A-state of cytochrome c, we monitored the changes in the 416 nm Cotton effect induced by increasing amounts of perchlorate and chloride.
• As shown in Fig. 6, addition of monovalent anions alters the 416 nm dichroic band; this suggests competition between monovalent and divalent anions for binding to the protein.
• In particular, both perchlorate and Cl shift the M-Fe(III)-H ↔ H-Fe(III)-H equilibrium towards the bis-H species, and destabilize the M-Fe(III)-H coordinated form.
• The reduced effect of Cl reflects the different affinities of the two anions for the protein [11,17].
• We also monitored the effect of sulfate on the perchlorate-induced A-state.
• As shown in Fig. 6, addition of sulfate strengthens the 416 nm dichroic band, which confirms that divalent anions have a greater tendency to stabilize the M80–Fe(III)-H18 coordinated form.
• On the whole, these data support competitive anion binding to the protein, and the idea that monovalent and divalent anions tend to stabilize differently structured A-states.
• Horse ferricytochrome c variants
• Anions carrying multiple negative charges bind to specific sites of horse cytochrome c [19,27].
• To determine whether divalent anions bind to the same sites, we introduced some mutations within the site-containing regions of the macromolecule, with the aim of defining the role played by single residues in modulating protein affinity for divalent anions.
• On the basis of earlier work [19,27], the sites under consideration were: (a) the site encompassing residues K87, K88, and R91, located in the C-terminal a-helix segment, indicated here as site 1; and (b) the site encompassing residues K86, K87, and K13, located at the interface between the N-terminal and the C-terminal a-helices, indicated here as site 2.
• The residues under investigation were substituted with residues located at the same position in yeast iso-1-cytochrome c; as illustrated in Fig. 7, horse and yeast cytochrome c show very different affinities (considered here as a nonspecific indicator of the binding effect, not as a direct measure of anion binding to the protein) for anions.
• CD and absorption measurements
• In site 1, the K88E mutation introduces an acidic residue (E88, present in yeast [28]) in place of a lysine, whereas in site 2, the K13N mutation introduces an asparagine in place of a lysine.
• This provides the opportunity to evaluate the contribution of K88 and K13 to protein stabilization in the reaction with sulfate.
• The far-UV and Soret CD spectra of the two mutants (not shown) reveal that the two variants and the wild-type protein are equally influenced by sulfate.
• Similar results were obtained when we investigated the spectroscopic properties of the K88E/T89K double mutant, which, with respect to the K88E mutant, possesses a sequence closer to the corresponding sequence in yeast iso-1-cytochrome c.
• A 40 mm sulfate concentration induced, in all the variants investigated, native-like a-helix content and formation of the 416 nm Cotton effect with a strength comparable (although not identical) to that of the wild-type protein.
• This excludes the possibility that K88, T89 and K13 modulate horse cytochrome c affinity for anions.
• Also, the mutant’s stability is not dissimilar to that of the wild-type protein, as indicated by thermal denaturation studies (data not shown).
• Fast kinetic measurements
• The 350-700 nm absorption spectrum of acid-denatured cytochrome c (spectrum a of Fig. 8A) displays an absorption maximum around 395 nm in the Soret region, and a maximum at 497 nm, a shoulder at 528 nm and a charge transfer (CT) at 618 nm in the visible region.
• The spectral changes detected at pH 2.2 upon mixing acid-denatured cytochrome c with sulfate (final anion concentration 40 mm) are shown in Fig. 8B.
• At 395 nm, the kinetic process appears to be biphasic, characterized by a fast phase (kobs = 50 ± 40 s⁻¹) and a slow phase (kobs = 8.4 ± 0.9 s⁻¹).
• The process is characterized by a red-shift of the Soret band (initially centered at 395 nm) to 402 nm.
• In the visible region, complex spectral changes are detected; in particular, the fast phase is characterized by a slight increase of the absorbance band centered at 528 nm and by a blue-shift of the CT band from 618 to 616 nm (spectrum b of Fig. 8A).
• The slow phase is instead characterized by a marked enhancement of the absorbance band centered at 528 nm (at the expense of the 497 nm peak), whereas the CT band decreases in intensity and red-shifts from 616 nm to 623 nm.
• This is also accompanied by an increase of the 695 nm band (spectrum c of Fig. 8A), with a rate close to that observed for the slow phase at 395 nm (Fig. 8B).
• Even though variations of the CT 623 nm band may contribute to the absorption change at 695 nm (thus affecting the amplitude change), the major contribution stems from the 695 nm band; therefore, the observed rate can be attributed to formation of the Fe(III)-M80 axial bond, providing strong indication that the slow phase is coupled to formation of the native axial coordination.
• The absorption spectra of the K13N and K88E/T89K mutants, shown in Fig. 9A, differ significantly from that of the acid-denatured cytochrome c.
• The S