Hydrophobic Interior Of Protein Research Articles

Different pKa Shifts of Internal GLU8 in Human β-Endorphin Amyloid Revealing a Coupling of Internal Ionization and Stepwise Fibril Disassembly.

As a functional amyloid, human β-endorphin amyloid fibril features a β-solenoid conformation and store peptide hormones within acidic secretory granules, which would be released into the blood through fibril disassembly when the cellular milieu pH increases from acidic to neutral level on exocytosis. To gain detailed atomic mechanism of β-endorphin amyloid fibrils' pH-responsive disassembly, we conduct constant pH molecular dynamics simulations to investigate the structural and dynamical properties of β-endorphin amyloid fibrils in experiencing the environmental pH changes. Our results demonstrate a clear pKa shift of the internal ionizable residue of GLU8, and this shift becomes even more pronounced when it is buried more deeply in the amyloid fibrils. The unusual pKa of GLU8 reveals that its protonation state changes from the protonated state in the acidic secretory granule to the deprotonated state in the neutral pH conditions in the blood, where the deprotonation of GLU8 leads to unfavorable interactions within the hydrophobic core of the amyloid and subsequent fibril disassembly. The different pKa shifts of GLU8 relative to its positions in the amyloid fibril indicate that the β-endorphin amyloid fibril disassembly is a stepwise process, accounting for the experimental observation that the disassembly always initiates from the outermost layer. This study reveals the critical role of the protonation state of GLU8 in amyloid fibrils' pH-responsive disassembly, and provides clear insights for understanding the structural transitions of amyloids in hormone secretion. This study also provides theoretical basis for designing pH-sensitive biological tools for specific use with precise positioning of ionizable residues into the hydrophobic interior of proteins.

Detection of simple proteins by direct surface-enhanced Raman scattering based on the Hofmeister ion-specific effect

Surface-enhanced Raman scattering (SERS) spectroscopy has unique advantages in detecting biomolecules, but label-free determination of proteins with low scattering cross-sections remains challenging. In this study, such proteins’ SERS signals have been optimized using the Hofmeister effect between protein molecules and CsI solution at physiological concentrations (A 100 mmol/L Cesium iodide, CsI). Cs+ as chaotro cation ion has a complex interaction mechanism with protein, can not only deprive hydrated water molecules on the surface of protein but also penetrate into the hydrophobic interior of protein. In addition to the above advantages, I- in excess CsI solution with appropriate concentration can removes the interference of citric acid-based impurities on the surface of silver nanoparticles, and Cs+ in excess CsI solution attracts the aggregation of negatively charged silver nanoparticles and cause local electromagnetic field enhancement to achieve high sensitivity in protein detection. This has been combined with principal component analysis to perform a comprehensive analysis of several proteins. Molecular dynamics simulations have been performed to study the mechanism of interaction between CsI and proteins. In addition, the vibrational peak of water has been used as an internal standard to quantify the protein content, and a good linear relationship between peak intensity and concentration was obtained.

Hydrostatic High-Pressure-Induced Denaturation of LH2 Membrane Proteins.

The denaturation of globular proteins by high pressure is frequently associated with the release of internal voids and/or the exposure of the hydrophobic protein interior to a polar aqueous solvent. Similar evidence with respect to membrane proteins is not available. Here, we investigate the impact of hydrostatic pressures reaching 12 kbar on light-harvesting 2 integral membrane complexes of purple photosynthetic bacteria using two types of innate chromophores in separate strategic locations: bacteriochlorophyll-a in the hydrophobic interior and tryptophan at both protein-solvent interfacial gateways to internal voids. The complexes from mutant Rhodobacter sphaeroides with low resilience against pressure were considered in parallel with the naturally robust complexes of Thermochromatium tepidum. In the former case, a firm correlation was established between the abrupt blue shift of the bacteriochlorophyll-a exciton absorption, a known indicator of the breakage of tertiary structure pigment-protein hydrogen bonds, and the quenching of tryptophan fluorescence, a supposed result of further protein solvation. No such effects were observed in the reference complex. While these data may be naively taken as supporting evidence of the governing role of hydration, the analysis of atomistic model structures of the complexes confirmed the critical part of the structure in the pressure-induced denaturation of the membrane proteins studied.

PH-Dependent Conformational Changes Lead to a Highly Shifted pKa for a Buried Glutamic Acid Mutant of SNase.

Ionizable residues are rarely present in the hydrophobic interior of proteins, but when they are, they play important roles in biological processes such as energy transduction and enzyme catalysis. Internal ionizable residues have anomalous experimental pKa values with respect to their pKa in bulk water. This work investigates the atomistic cause of the highly shifted pKa of the internal Glu23 in the artificially mutated variant V23E of Staphylococcal Nuclease (SNase) using pH replica exchange molecular dynamics (pH-REMD) simulations. The pKa of Glu23 obtained from our calculations is 6.55, which is elevated with respect to the glutamate pKa of 4.40 in bulk water. The calculated value is close to the experimental pKa of 7.10. Our simulations show that the highly shifted pKa of Glu23 is the product of a pH-dependent conformational change, which has been observed experimentally and also seen in our simulations. We carry out an analysis of this pH-dependent conformational change in response to the protonation state change of Glu23. Using a four-state thermodynamic model, we estimate the two conformation-specific pKa values of Glu23 and describe the coupling between the conformational and ionization equilibria.

Ion Pairs Buried in Hydrophobic Environments within Proteins: Electrostatic Crosstalk between Buried Groups

Internal ion pairs buried in the hydrophobic interior of proteins are essential for processes such as H+ transport, e- transfer, and catalysis. Despite their importance, the properties of buried ion pairs remain poorly understood. It has been suggested that medium- or long-range Coulomb interactions could play a role in tuning biological function, but this would only be possible if proteins behaved as materials with low dielectric constant, as assumed in many continuum electrostatics models. Buried ion pairs are of interest then not only for their functional roles, but also because they can be used to examine the balance between Coulomb and hydration energies in the dry, hydrophobic interior of proteins. As the distance between a pair of internal ionizable groups increases and the energy of their Coulomb interaction decreases, the balance of these energies will favor burial of the groups in the neutral state, eliminating any direct electrostatic interaction between them. To examine the magnitude of electrostatic crosstalk between buried ionizable groups experimentally, many variants were engineered on a highly stable form of staph nuclease. All variants have His66 in the hydrophobic core and either Asp or Glu residues buried at another internal position. Many His-Asp/Glu pairs were studied, and in no case were medium- or long-range Coulomb interactions observed. When the groups were within H-bond distance, strong, favorable coupling energies were measured, but only when the side chains of the buried pair could achieve a geometry favorable for an H-bond interaction. These data suggest that at short range the buried pair behaved as a strong hydrogen bond, but no evidence was found that they ever behave as titratable point charges interacting at a distance through Coulomb forces enhanced by the low dielectric protein medium.

A Coupled Ionization-Conformational Equilibrium Is Required To Understand the Properties of Ionizable Residues in the Hydrophobic Interior of Staphylococcal Nuclease.

Ionizable residues in the interior of proteins play essential roles, especially in biological energy transduction, but are relatively rare and seem incompatible with the complex and polar environment. We perform a comprehensive study of the internal ionizable residues on 21 variants of staphylococcal nuclease with internal Lys, Glu, or Asp residues. Using pH replica exchange molecular dynamics simulations, we find that, in most cases, the pKa values of these internal ionizable residues are shifted significantly from their values in solution. Our calculated results are in excellent agreement with the experimental observations of the Garcia-Moreno group. We show that the interpretation of the experimental pKa values requires the study of not only protonation changes but also conformational changes. The coupling between the protonation and conformational equilibria suggests a mechanism for efficient pH-sensing and regulation in proteins. This study provides new physical insights into how internal ionizable residues behave in the hydrophobic interior of proteins.

Local Backbone Flexibility as a Determinant of the Apparent pKa Values of Buried Ionizable Groups in Proteins.

Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly anomalous pKa values that reflect the incompatibility between charges and dehydrated environments. A systematic study of pKa values of buried ionizable groups in staphylococcal nuclease (SNase) suggests that these pKa values are determined in part by conformational reorganization of the protein. Lys-66 is one of the most deeply buried residues in SNase. We show that its apparent pKa of 5.7 reflects the average of the pKa values of Lys-66 in different conformational states of the protein. In the fully folded state, Lys-66 is deeply buried in the hydrophobic core of SNase and must titrate with a pKa of ≪5.7. In other states, the side chain of Lys-66 is fully solvent-exposed and has a normal pKa of ≈10.4. We show that the pKa of Lys-66 can be shifted from 5.7 toward a more normal value of 7.1 via the insertion of flanking Gly residues at positions 64 and 67 to promote an "open" conformation of SNase. Crystal structures and nuclear magnetic resonance spectroscopy show that in these Gly-containing variants Lys-66 can access bulk water as a consequence of overwinding of the C-terminal end of helix 1. These data illustrate that the apparent pKa values of buried groups in proteins are governed in part by the difference in free energy between different conformational states of the protein and by differences in the pKa values of the buried groups in the different conformations.

A General Mechanism for the Propagation of Mutational Effects in Proteins.

Mutations in the hydrophobic interior of proteins are generally thought to weaken the interactions only in their immediate neighborhood. This forms the basis of protein engineering-based studies of folding mechanism and function. However, mutational work on diverse proteins has shown that distant residues are thermodynamically coupled, with the network of interactions within the protein acting as signal conduits, thus raising an intriguing paradox. Are mutational effects localized, and if not, is there a general rule for the extent of percolation and the functional form of this propagation? We explore these questions from multiple perspectives in this work. Perturbation analysis of interaction networks within proteins and microsecond long molecular dynamics simulations of several aliphatic mutants of ubiquitin reveal strong evidence of the distinct alteration of distal residue-residue communication networks. We find that mutational effects consistently propagate into the second shell of the altered site (even up to 15-20 Å) in proportion to the perturbation magnitude and dissipate exponentially with a decay distance constant of ∼4-5 Å. We also report evidence for this phenomenon from published experimental nuclear magnetic resonance data that strikingly resemble predictions from network theory and molecular dynamics simulations. Reformulating these observations onto a statistical mechanical model, we reproduce the stability changes of 375 mutations from 19 single-domain proteins. Our work thus reveals a robust energy dissipation-cum-signaling mechanism in the interaction network within proteins, quantifies the partitioning of destabilization energetics around the mutation neighborhood, and presents a simple theoretical framework for modeling the allosteric effects of point mutations.

Conformational Reorganization Coupled to the Ionization of Internal Lys Residues in Proteins.

Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction and catalysis. Because the protein interior is usually neither as polar nor as polarizable as water, these groups tend to have anomalous pKa values, and their ionization tends to be coupled to conformational reorganization. To elucidate mechanisms of energy transduction in proteins, it is necessary to understand the structural determinants of the pKa values of these buried groups, including the range and character of the conformational reorganization that the ionization of these buried groups can elicit. The L25K and L125K variants of staphylococcal nuclease (SNase) were used to characterize the diverse types of structural reorganization that can be promoted by the ionization of buried groups. NMR relaxation dispersion and ZZ-exchange experiments were used to identify the locations and measure the time scales and extent of pH-dependent conformational exchange in these two proteins. The buried Lys-25 and Lys-125 residues titrate with pKa of 6.3 and 6.2, respectively. The L25K protein fluctuates between the native state and an ensemble of locally unfolded states on the 400 μs to 7 ms time scale. On the 100 to 500 ms time scale the native state exchanges with a subglobally unfolded state in which the β-barrel is partially reorganized. The equilibrium between the native state and this alternative state is highly pH dependent; at pH values below the pKa of Lys-25 the state with the partially reorganized β-barrel is the dominant state. In contrast, the L125K protein only exhibited pH-independent fluctuation in the microsecond to millisecond time scale in the region near Lys-125. The study illustrates how diverse and how localized the coupling between conformational reorganization and ionization of buried groups can be. The pH-sensitive exchange between the fully native and subglobally or locally unfolded states in time scales well into hundreds of milliseconds will challenge all computational methods for structure-based calculations of pKa values.

Interactions between Pairs of Charges Buried in the Hydrophobic Interior of a Protein are Unexpectedly Weak

Internal ion pairs buried in the hydrophobic protein interior are essential for many important biochemical processes, including H+ transport, e- transfer, ion homeostasis, and catalysis. Despite the importance of these pairs, their properties remain poorly understood. It has been suggested that, for some systems, medium or long-range Coulomb interactions between buried groups could play a role in determining biological function. In principle, this should only be possible if the protein interior behaves like a medium of low dielectric constant, as assumed in most electrostatics models. These motifs are then of special interest not only for their functional roles, but also as probes to examine the balance between Coulomb and hydration energies experienced by buried charges. As the distance between the internal charges increases, the balance of these energies will disfavor burying the groups in the charged state, eliminating any Coulomb interaction between them. To examine the balance between Coulomb and dehydration energies in the protein interior we engineered a series of double variants in a highly stable variant of staphylococcal nuclease. Each variant included an internal histidine paired with either an aspartate or glutamate buried at various internal positions throughout the protein interior. This set of proteins used to probe the distance dependence of potential Coulomb interactions. No significant electrostatic coupling was observed for pairs that interacted through medium- or long-range Coulomb interactions. When the distance between the groups was short, favorable coupling energies were only observed when the side chains of the pair could achieve a geometry favorable for a H-bond; spatial proximity alone was insufficient to create favorable Coulomb interactions. Simple electrostatics models that describe ionized states as point charges interacting through space are unlikely to be able to reproduce these data.

Structural Reorganization Triggered by the Ionization of Lys Residues buried in Hydrophobic Environments

A study of the structural consequences of ionization of amino acid side chains buried in the hydrophobic interior of proteins was performed by systematic introduction of lysine at 25 internal positions in a highly stable form of staphylococcal nuclease. Crystal structures for 12 of the variants at pH values where the Lys was expected to be uncharged revealed nearly identical backbones with the reference protein and ordered internal Lys side chains at the substitution site. The structures demonstrate that neutral Lys can be buried in the protein interior without the need for any specialized structural adaptations save for those required to stabilize the protein. Solvent-accessible surface area and depth of burial calculations indicated that all but one Lys were completely inaccessible to water. Seven Lys side chains packed into predominantly hydrophobic regions; five had Nζ atoms within 3.5 A of one or more polar groups or crystallographic water molecules. Depth of burial or microenvironment polarity as observed in the crystal structures did not correlate with measured shifts in pKa. 1H-15N HSQC NMR spectroscopy studies revealed global unfolding coupled to the ionization of two of the 25 Lys residues. Three others showed partial or local unfolding, five demonstrated localized changes in structure and dynamics, and 15 exhibited no detectable consequences. Backbone amide resonances were most similar between reference protein and the Lys-containing variant when the Lys side chains were buried shallowly or in a loop region. In the least stable variants the ionization of the internal Lys residues led to localized increase in conformational fluctuations or to conformational reorganization. These data will be useful to test the ability of structure-based pKa calculations to account for structural rearrangements or increased dynamics in response to ionization of internal groups.

Charges in Hydrophobic Environments in Proteins

Ionizable groups buried in the hydrophobic interior of proteins play central roles in a wide variety of essential biochemical processes. Owing to the inherent differences in the dielectric properties of water and of protein, buried ionizable groups usually titrate with anomalous pKa values shifted relative to normal pKa values in water in the direction that favors the neutral state. The determinants of these unusual pKa values are being examined systematically with a family of variants of staphylococcal nuclease with Lys, Arg, His, Asp and Glu in 25 internal positions. Some of these ionizable groups titrate with normal pKa values, but the majority of them have anomalous pKa values, some shifted by as many as 6 units relative to the normal values. Dozens of crystal structures confirm that the ionizable groups are buried, at least when they are in the neutral state, often in complex with water molecules never before observed at internal locations in SNase. The structures show that the microenvironments of the ionizable groups range between the fully hydrophobic to the highly polar, but they do not fully explain the unusual properties of these groups. NMR spectroscopy has been used to examine the range of structural reorganization that can be coupled to the ionization of the internal ionizable. While the ionization of some residues is not easily detected in HSQC spectra, in most cases there is clear evidence of modest charge-induced changes in local structure or increased conformational fluctuations. Interactions between internal groups and polar groups, surface charges, and other internal groups have also been measured. These data constitute an extensive, quantitative description of origins of the apparent dielectric properties of proteins. They also show that proteins tolerate internal charges without the need for any special structural adaptations.

Identification and Characterization of Buried Ionizable Groups in Proteins

Ionizable groups buried in the hydrophobic interior of proteins play many essential biological roles. their physical properties are not well understood. Owing to the inherent differences in the dielectric properties of proteins and of water the properties of internal and surface ionizable groups can be very different. In a previous systematic study from this laboratory Asp, Glu, Lys and Arg residues were buried at 25 internal locations in staphylococcal nuclease. Most of these internal residues titrate with highly anomalous pKa values. Many crystal structures of these variants show that the majority of these buried ionizable residues are buried completely and in very diverse microenvironments. A survey of the properties and microenvironments of naturally occurring internal ionizable groups in proteins was undertaken with a large set of proteins from the Protein Data Bank to compare against the internal ionizable groups buried artificially in nuclease. Internal ionizable groups were identified based on accessible surface area and depth of burial using a self-consistent definition of the interface between protein and bulk water. For purposes of this survey, internal ionizable groups were characterized primarily in terms of accessible surface area, depth of burial, polarity and hydrophobicity of their microenvironments, and contact with other surface or internal ionizable groups and with crystallographic water molecules. MD simulations were also performed on a small set of proteins to examine the robustness of static structures for purposes of determination of depth of burial.

Effect of trimethylamine-N-oxide on pressure-induced dissolution of hydrophobic solute

Molecular dynamics simulations are performed to study the effects of increasing trimethylamine-N-oxide (TMAO) concentration on the pressure-induced dissolution of hydrophobic solutes immersed in water. Such systems are of interest mainly because pressure increases the dissolution of hydrophobic protein interior causing protein denaturation and TMAO acts to offset the protein denaturing effect of high hydrostatic pressures. In view of this, in this study, methane molecules are considered as model hydrophobic molecules and simulations are performed for four independent TMAO solutions each at four different pressures ranging from 2 to 8 kbar. From potentials of mean force calculations, it is found that application of pressure reduces the free energy difference between contact minimum (CM) and solvent-separated (SSM) minimum of hydrophobic solute, suggesting dissolution at high pressures. TMAO, on the other hand, increases the relative stability of CM state of methane molecules relative to its SSM state. High packing efficiency of water molecules around the hydrophobic solute at high pressure is observed. Also observed are TMAO-induced enhancement of water structure and direct hydrogen-bonding interaction between TMAO and water and the correlated dehydration of hydrophobic solute. From hydrogen bond properties and dynamics calculations, it is observed that pressure increases average number of water-water hydrogen bonds while reduces their life-times. In contrast, TMAO reduces water-water hydrogen bonding but enhances their life-times. These results suggest that TMAO can reduce water penetration into the protein interior by enhancing water structure and also forming hydrogen bonds with water and hence counteracts protein unfolding.

Potential charge transfer probe induced conformational changes of model plasma protein human serum albumin: Spectroscopic, molecular docking, and molecular dynamics simulation study

The nature of binding of specially designed charge transfer (CT) fluorophore at the hydrophobic protein interior of human serum albumin (HSA) has been explored by massive blue-shift (82 nm) of the polarity sensitive probe emission accompanying increase in emission intensity, fluorescence anisotropy, red edge excitation shift, and average fluorescence lifetimes. Thermal unfolding of the intramolecular CT probe bound HSA produces almost opposite spectral changes. The spectral responses of the molecule reveal that it can be used as an extrinsic fluorescent reporter for similar biological systems. Circular dichrosim spectra, molecular docking, and molecular dynamics simulation studies scrutinize this binding process and stability of the protein probe complex more closely.

Structural Reorganization Triggered by Charging of Lys Residues in the Hydrophobic Interior of a Protein

Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK(a) calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.

Study of microheterogeneous environment of protein Human Serum Albumin by an extrinsic fluorescent reporter: A spectroscopic study in combination with Molecular Docking and Molecular Dynamics Simulation

We report extrinsic fluorescent probe 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) as a molecular reporter for studying microheterogeneous environment of protein Human Serum Albumin (HSA) via spectral modification of the probe under physiological condition. Steady state emission, fluorescence anisotropy, Red Edge Excitation Shift (REES), far-UV Circular Dichroism (CD), Atomic Force Microscopy (AFM) imaging, time resolved spectral measurements, Molecular Docking and Molecular Dynamics (MD) Simulation techniques have been used to fulfill this achievement. Interaction of the probe with HSA is signaled by the blue shift of the fluorophore emission maxima with enhancement of fluorescence intensity. The increase in steady state anisotropy, REES and fluorescence lifetime values with increasing protein concentrations indicates interaction and movement of the probe from free aqueous media to the more restricted less polar hydrophobic interior of protein. Experimental results obtained from Benesi–Hildebrand plot support the formation of 1:1 HSA–DMAPPDA complex with high binding constant and negative free energy change. Thermal denaturation of the probe bound protein has also been tracked using the spectral response of DMAPPDA. Molecular Docking studies revealed binding of the probe with in the hydrophobic cavity of subdomain IIA of HSA. MD Simulation supports greater stability of HSA–DMAPPDA complex compared to free protein.

Extended protein/water H-bond networks in photosynthetic water oxidation

Oxidation of water molecules in the photosystem II (PSII) protein complex proceeds at the manganese–calcium complex, which is buried deeply in the lumenal part of PSII. Understanding the PSII function requires knowledge of the intricate coupling between the water-oxidation chemistry and the dynamic proton management by the PSII protein matrix. Here we assess the structural basis for long-distance proton transfer in the interior of PSII and for proton management at its surface. Using the recent high-resolution crystal structure of PSII, we investigate prominent hydrogen-bonded networks of the lumenal side of PSII. This analysis leads to the identification of clusters of polar groups and hydrogen-bonded networks consisting of amino acid residues and water molecules. We suggest that long-distance proton transfer and conformational coupling is facilitated by hydrogen-bonded networks that often involve more than one protein subunit. Proton-storing Asp/Glu dyads, such as the D1-E65/D2-E312 dyad connected to a complex water-wire network, may be particularly important for coupling protonation states to the protein conformation. Clusters of carboxylic amino acids could participate in proton management at the lumenal surface of PSII. We propose that rather than having a classical hydrophobic protein interior, the lumenal side of PSII resembles a complex polyelectrolyte with evolutionary optimized hydrogen-bonding networks. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Arginine residues at internal positions in a protein are always charged

Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK(a) values can be highly anomalous, some shifted by as many as 5.7pH units relative to normal pK(a) values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK(a); they are all charged at pH≤10. Twenty-three of these 25 variants with Arg are folded at both pH7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments.

How many ionizable groups can sit on a protein hydrophobic core?

Full or partial burial of ionizable groups in the hydrophobic interior of proteins underlies the large modulation in group properties (modified pK value, high nucleophilicity, enhanced capability of interaction with chemical moieties of the substrate, etc.) linked to biological function. Indeed, the few internal ionizable residues found in proteins are known to play important functional roles in catalysis and, in general, in energy transduction processes. However, ionizable-group burial is expected to be seriously disruptive and, it is important to note, most functional sites contain not just one, but several ionizable residues. Hence, the adaptations involved in the development of function in proteins (through in vitro engineering or during the course of natural evolution) are not fully understood. Here, we explore experimentally how proteins respond to the accumulation of hydrophobic-to-ionizable residue substitutions. For this purpose, we have constructed a combinatorial library targeting a hydrophobic cluster in a consensus-engineered, stabilized form of a small model protein. Contrary to naïve expectation, half of the variants randomly selected from the library are soluble, folded, and active, despite including up to four mutations. Furthermore, for these variants, the dependence of stability with the number of mutations is not synergistic and catastrophic, but smooth and approximately linear. Clearly, stabilized protein scaffolds may be robust enough to withstand many disruptive hydrophobic-to-ionizable residue mutations, even when they are introduced in the same region of the structure. These results should be relevant for protein engineering and may have implications for the understanding of the early evolution of enzymes.