Hydrophobic Protein Environment Research Articles

Charges in hydrophobic environments in proteins: A study of arginine ion-pairs

Charges in hydrophobic environments in proteins: A study of arginine ion-pairs

Thermodynamic stability as a determinant of the pKa values of ionizable residues in hydrophobic environments in proteins

Thermodynamic stability as a determinant of the pKa values of ionizable residues in hydrophobic environments in proteins

Anomalous Properties of Lys Residues Buried in Hydrophobic Environments in Proteins

Anomalous Properties of Lys Residues Buried in Hydrophobic Environments in Proteins

Anomalous Properties of Lys Residues Buried in the Hydrophobic Interior of a Protein Revealed with 15N-Detect NMR Spectroscopy.

Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pKa values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues that titrate with pKa values ranging from 6.2 to 8.1. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how conformational transitions coupled to their ionization affect their pKa values.

Factors Determining the Rate and Selectivity of 4e-/4H+ Electrocatalytic Reduction of Dioxygen by Iron Porphyrin Complexes.

Reactivity as well as selectivity are crucial in the activation and electrocatalytic reduction of molecular oxygen. Recent developments in the understanding of the mechanism of electrocatalytic O2 reduction by iron porphyrin complexes in situ using surface enhanced resonance Raman spectroscopy coupled to rotating disc electrochemistry (SERRS-RDE) in conjunction with H/D isotope effects on electrocatalytic current reveals that the rate of O2 reduction, ∼104 to 105 M-1 s-1 for simple iron porphyrins, is limited by the rate of O-O bond cleavage of an intermediate ferric peroxide species (FeIII-OOH). SERRS-RDE probes the system in operando when it is under steady state such that any intermediate species that has a greater rate of formation relative to its rate of decay, including the rate determining species, would accumulate and can be identified. This technique is particularly well suited to investigate iron porphyrin electrocatalysts as the intense symmetric ligand vibrations allow determination of the oxidation and spin states of the bound iron with high fidelity. The rate of O2 reduction could be tuned up by 3 orders of magnitude by incorporating residues in the catalyst design that can exert "push" or "pull" effects, that is, axial phenolate and thiolate ligands and distal arginine residues. Similarly the rate of O-O bond cleavage can be enhanced by several orders of magnitude upon incorporating a distal Cu site and installing the active site in a hydrophobic protein environment in synthetic models and biosynthetic protein scaffolds. The selectivity, however, is solely determined by the site of protonation of a ferric peroxide (FeIII-OOH) intermediate and can be governed by installing preorganized second sphere residues in the distal pocket. The 4e-/4H+ reduction of O2 entails protonation of the distal oxygen of the FeIII-OOH species, while 2e-/2H+ reduction requires the proximal oxygen to be protonated. Mechanistic investigations of CO2 reduction by iron porphyrins reveal that the rate-determining step is the C-O bond cleavage of a FeII-COOH species analogous to the O-O bond cleavage step of a FeIII-OOH species in O2 reduction. The selectivity, resulting in either CO or HCOOH, is determined by the site of protonation of this species. These similarities suggests that the chemical principles governing the rate and selectivity of reduction of small molecules like O2, CO2, NOx, and SOx may be quite similar in nature.

Proteins as pH Sensors and Switches

Ionizable residues buried in dry or hydrophobic environments in proteins can have extremely anomalous pKa values (pKa > 10 for Asp and Glu, pKa 5 for Lys). These buried ionizable groups are involved in many forms of biological energy transduction. Their anomalous pKa values are usually essential for function in many pH-driven processes. Anomalous pKa values impart unusual pH sensitivity to the stability of the folded state. Previously we examined energetic consequences of burial of Lys, His, Arg, Asp and Glu in hydrophobic environments systematically, using staphylococcal nuclease (SNase) as a model system. With NMR spectroscopy and X-ray crystallography we have now characterized a variety of different types of conformational reorganization coupled to the ionization of these buried groups. Not only are conformational transitions driven by the binding or release of an H+ the basis of many H+-coupled energy transduction processes (e.g. ATPase), the detailed structural characterization of locally, partially or subglobally unfolded forms of SNase has also contributed a unique perspective on its folding landscape. When they occur, these structural transitions determine the magnitude of the shift in the pKa of internal groups. We have now also demonstrated that the unusual properties of buried ionizable groups can be harnessed to engineer proteins capable of acting as pH sensors and pH-driven switches. These switch proteins are sensitive to changes in pH in the physiological range despite using Asp, Glu or Lys residues for purposes of pH sensing. I will describe specific examples illustrating general and transferable engineering principles that may be used to turn any protein into a pH-sensitive protein with structure or function that can be modulated by small changes in pH in the physiological range.

Conformational Exchange Coupled to Ionization of Lys Residues Buried in Hydrophobic Environments in Proteins

Ionizable amino acids buried in hydrophobic environments in proteins can titrate with anomalous pKa values. Despite the central role of these amino acids in energy transduction and catalysis, the determinants of their unusual properties are difficult to reproduce with computational approaches. Conformational reorganization of the protein backbone is a key determinant of pKa values. This reflects the ability of the protein to sample conformational microstates, which is particularly challenging to reproduce computationally. To characterize structural details and timescales of conformational reorganization coupled to ionization of buried groups, NMR spectroscopy was used to identify locations, measure timescales, and determine the extent of pH dependence of conformational exchange in the L25K and L125K variants of staphylococcal nuclease. Lys-25 or Lys-125 titrate with pKa values of 6.3 and 6.2, respectively. Using relaxation dispersion spectroscopy we observed that the β barrel of the L25K protein fluctuates between the native and an alternate state on the 0.4 to 7 ms timescale. The HN chemical shifts of the alternate state, assigned using ZZ-exchange spectroscopy, were less dispersed than those of the native state, consistent with unfolding of the β barrel. The equilibrium between these states is strongly pH dependent, with the partially unfolded state dominating at pH values at which the internal Lys residue is charged. In contrast, the L125K protein exhibited fluctuations near Lys-125 on the µs-ms timescale but in a faster regime than the L25K protein and without pH dependence. These results show the extent of coupling between conformational change and proton binding is highly dependent on local structural properties. Computational models attempting to reproduce the properties and pKa values of buried ionizable groups must be able to represent conformational exchange between native and subglobally unfolded states on ms timescales.

PKA Values of Buried Groups in Proteins are Sensitive to the Global Thermodynamic Stability

Ionizable groups buried in hydrophobic environments in proteins are essential for all forms of biological energy transduction. The molecular determinants of the pKa values of these internal groups are poorly understood. It is increasingly apparent that conformational reorganization coupled to the ionization of the buried group is a major determinant of these pKa values. Specifically, the creation of charge in hydrophobic environments can trigger a shift from the fully folded state to local or partially unfolded states in which the charge can gain access to water or to an environment where the charge can be solvated. These alternative conformational states are not normally populated owing to the large free energy difference between the alternative and fully-folded native states; however, the partially unfolded states can become the new ground state under pH conditions where the internal group is charged. If the ionization of an internal group promotes the transition to a new conformational state then its pKa should be sensitive to the global thermodynamic stability (ΔG°) of the protein because this determines the energy gap between the ground and the alternative states. This was tested by measuring the pKa of two internal Lys residues in variants of staphylococcal nuclease with thermodynamic stabilities ranging from 8.4 to 13.8 kcal/mol. The magnitude of the shift in the pKa of the internal Lys residues was found to be sensitive to the ΔG° of the protein confirming that the pKa values of these Lys residues are determined by the probability of structural reorganization more than by local dielectric properties of their microenvironments. These observations imply that structure-based pKa calculations for buried groups and other electrostatic processes in hydrophobic environments require accurate treatment of conformational reorganization, which remains an extremely challenging proposition.

Backbone Flexibility as a Determinant of PKA Values of Buried Ionizable Groups in Proteins

Ionizable groups buried in hydrophobic environments in proteins play essential roles in biological energy transduction. These buried groups usually titrate with anomalous pKa values, shifted in the direction that favors the neutral state because charges are not compatible with the dehydrated interior of proteins. Owing to their importance in catalysis, H+ transport and other forms of biological energy transduction, the molecular determinants of these pKa values are of interest. In particular, the extent to which flexibility of the backbone contributes to the apparent pKa values measured experimentally is not well understood. In flexible regions the ionizable groups can sample different microenvironments with different dielectric properties. The apparent pKa values measured reflect an ensemble-weighted average of the true pKa values in the different microenvironments. To examine the role of backbone flexibility we attempted to modulate the pKa of the internal Lys-66 and Lys-25 in staphylococcal nuclease through mutations of selected positions to Gly to increase the flexibility of the backbone. The hypothesis that was tested posits that an increase in flexibility would shift the anomalous pKa values of these Lys residues (5.7 for Lys-66 and 7.1 for Lys-25) towards more normal values near 10.4 because the removal of conformational restrictions on the backbone would increase the population of conformational states in which the previously buried ionizable group would become more exposed to water, and this would lead to more normal pKa normal values. Besides demonstrating that pKa values are affected by substitutions to Gly in the manner expected, structural methods corroborated that an increase in apparent pKa was the result of increased exposure of the Lys side chains to water.

Spectral and functional studies on siphonaxanthin-type light-harvesting complex of photosystem II from Bryopsis corticulans

Carotenoids with conjugated carbonyl groups possess special photophysical properties which have been studied in some water-soluble light-harvesting proteins (Polívka and Sundström, Chem Rev 104:2021-2071, 2004). However, siphonaxanthin-type light-harvesting complexes of photosystem II (LHCII) in siphonous green alga have received fewer studies. In the present study, we determined sequences of genes for several Bryopsis corticulans Lhcbm proteins, which showed that they belong to the group of major LHCII and diverged early from green algae and higher plants. Analysis of pigment composition indicated that this siphonaxanthin-type LHCII contained in total 3 siphonaxanthin and siphonein but no lutein and violaxanthin. In addition, 2 chlorophylls a in higher plant LHCII were replaced by chlorophyll b. These changes led to an increased absorption in green and blue-green light region compared with higher plant LHCII. The binding sites for chlorophylls, siphonaxanthin, and siphonein were suggested based on the structural comparison with that of higher plant LHCII. All of the ligands for the chlorophylls were completely conserved, suggesting that the two chlorophylls b were replaced by chlorophyll a without changing their binding sites in higher plant LHCII. Comparisons of the absorption spectra of isolated siphonaxanthin and siphonein in different organic solutions and the effect of heat treatment suggested that these pigments existed in a low hydrophobic protein environment, leading to an enhancement of light harvesting in the green light region. This low hydrophobic protein environment was maintained by the presence of more serine and threonine residues in B. corticulans LHCII. Finally, esterization of siphonein may also contribute to the enhanced harvesting of green light.

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.

Dke1—structure, dynamics, and function: a theoretical and experimental study elucidating the role of the binding site shape and the hydrogen-bonding network in catalysis

This study elucidates the role of the protein structure in the catalysis of β-diketone cleavage at the three-histidine metal center of diketone cleaving enzyme (Dke1) by computational methods in correlation with kinetic and mutational analyses. Molecular dynamics simulations, using quantum mechanically deduced parameters for the nonheme Fe(II) cofactor, were performed and showed a distinct organization of the hydrophilic triad in the free and substrate-ligated wild-type enzyme. It is shown that in the free species, the Fe(II) center is coordinated to three histidines and one glutamate, whereas the substrate-ligated, catalytically competent enzyme-substrate complex has an Fe(II) center with three-histidine coordination, with a small fraction of three-histidine, one-glutamate coordination. The substrate binding modes and channels for the traffic of water and ligands (2,4-pentandionyl anion, methylglyoxal, and acetate) were identified. To characterize the impact of the hydrophobic protein environment around the metal center on catalysis, a set of hydrophobic residues close to the active site were targeted. The variations resulted in an up to tenfold decrease of the O(2) reduction rates for the mutants. Molecular dynamics studies revealed an impact of the hydrophobic residues on the substrate stabilization in the active site as well as on the orientations of Glu98 and Arg80, which have previously been shown to be crucial for catalysis. Consequently, the Glu98-His104 interaction in the variants is weaker than in the wild-type complex. The role of protein structure in stabilizing the primary O(2) reduction step in Dke1 is discussed on the basis of our results.

Probing the Ca2+ Switch of the Neuronal Ca2+ Sensor GCAP2 by Time-Resolved Fluorescence Spectroscopy

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.

Electrostatic pKa computations in proteins: Role of internal cavities

The solvent accessible surface area (SASA) algorithm is conventionally used to characterize protein surfaces in electrostatic energy computations of proteins. Unfortunately, it often fails to find narrow cavities inside a protein. As a consequence pK(a) computations based on this algorithm perform badly. In this study a new cavity-algorithm is introduced, which solves this problem and provides improved pK(a) values. The procedure is applied to 20 pK(a) values of titratable groups introduced as point mutations in SNase variants, where crystal structures are available. The computations of these pK(a)s are particular challenging, since they are placed in a rather hydrophobic environment. For nine mutants, where the titratable residue is in contact with a large cavity, the RMSD(pKa) between computed and measured pK(a) values is 2.04, which is a considerable improvement as compared to the original results obtained with Karlsberg(+) (http://agknapp.chemie.fu-berlin.de/karlsberg/) that yielded an RMSD(pKa) of 8.8. However, for 11 titratable residues the agreement with experiments remains poor (RMSD(pKa) = 6.01). Considering 15 pK(a)s of SNase, which are in a more conventional less hydrophobic protein environment, the RMSD(pKa) is 2.1 using the SASA-algorithm and 1.7 using the new cavity-algorithm. The agreement is reasonable but less good than what one would expect from the general performance of Karlsberg(+) indicating that SNase belongs to the more difficult proteins with respect to pK(a) computations. We discuss the possible reasons for the remaining discrepancies between computed and measured pK(a)s.

Chemically Induced Unfolding of Bovine Serum Albumin by Urea and Sodium Dodecyl Sulfate: A Spectral Study with the Polarity‐Sensitive Charge‐Transfer Fluorescent Probe (E)‐3‐(4‐Methylaminophenyl)acrylic Acid Methyl Ester

Sensitivity of the charge-transfer (CT) band of the fluorescence probe (E)-3-(4-methylaminophenyl)acrylic acid methyl ester (MAPAME) towards the polarity of its immediate environment is employed to investigate the binding interaction of the probe with bovine serum albumin (BSA) and uncoiling of BSA by the denaturants urea and sodium dodecyl sulfate micelles. Binding of the probe with BSA produces a blue shift and enhanced intensity of the CT emission band which clearly point toward a decrease in polarity of the immediate environment of MAPAME. This is expected, since binding with BSA moves the probe from a polar water environment to a much less polar, hydrophobic protein interior, where the CT band is expected to be blue-shifted. Higher intensity arises due to fewer non-radiative decay paths available to the probe in the hydrophobic protein environment. Chemically induced unfolding of BSA by urea and sodium dodecyl sulfate is tracked by monitoring the induced spectral changes of the protein-bound probe MAPAME. Red-edge excitation shift or REES, fluorescence resonance energy transfer (FRET) and anisotropy measurements are used to investigate and monitor these binding and unfolding processes.

Assessing the Role of the Active-site Cysteine Ligand in the Superoxide Reductase from Desulfoarculus baarsii

Superoxide reductase is a novel class of non-heme iron proteins that catalyzes the one-electron reduction of O(2)(.) to H(2)O(2), providing an antioxidant defense in some bacteria. Its active site consists of an unusual non-heme Fe(2+) center in a [His(4) Cys(1)] square pyramidal pentacoordination. In this class of enzyme, the cysteine axial ligand has been hypothesized to be an essential feature in the reactivity of the enzyme. Previous Fourier transform infrared spectroscopy studies on the enzyme from Desulfoarculus baarsii revealed that a protonated carboxylate group, proposed to be the side chain of Glu(114), is in interaction with the cysteine ligand. In this work, using pulse radiolysis, Fourier transform infrared, and resonance Raman spectroscopies, we have investigated to what extent the presence of this Glu(114) carboxylic lateral chain affects the strength of the S-Fe bond and the reaction of the iron active site with superoxide. The E114A mutant shows significantly modified pulse radiolysis kinetics for the protonation process of the first reaction intermediate. Resonance Raman spectroscopy demonstrates that the E114A mutation results in both a strengthening of the S-Fe bond and an increase in the extent of freeze-trapping of a Fe-peroxo species after treatment with H(2)O(2) by a specific strengthening of the Fe-O bond. A fine tuning of the strength of the S-Fe bond by the presence of Glu(114) appears to be an essential factor for both the strength of the Fe-O bond and the pK(a) value of the Fe(3+)-peroxo intermediate species to form the reaction product H(2)O(2).

Binding of the fluorescent substrate analog 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5'-triphosphate to the gastric hydrogen ion-potassium-ATPase: evidence for cofactor-induced conformational changes in the enzyme

TNP-ATP binds to the gastric H,K-ATPase with a 4.6-fold increase in fluorescence intensity and 10-nm blue shift that indicate a relatively hydrophobic protein environment. The fluorescence enhancement saturates and is compatible with binding to a single class of specific nucleotide sites with Kd less than 25 nM and N = 3.4 +/- 0.9 nmol mg-1. Cofactors of the H,K-ATPase affect the fluorescence enhancement. K+ causes a rapid fluorescence quench by binding to a single class of sites with Kd = 3 mM. Mg2+ rapidly and completely reverses the K+ quench and then causes a slow fluorescence quench. The maximum enhancement is approximately halved by either Mg2+ or K+ in titrations with both protein and fluorophore. Therefore, TNP-ATP reports changes in protein environment compatible with cofactor-induced changes in the conformation of the enzyme.

Structure-Function Relationships in Lactate Dehydrogenase

The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate.