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1.
Biochemistry ; 56(1): 212-218, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28009501

RESUMO

In the V23E variant of staphylococcal nuclease, Glu-23 has a pKa of 7.5. At low pH, Glu-23 is neutral and buried in the hydrophobic interior of the protein. Crystal structures and NMR spectroscopy experiments show that when Glu-23 becomes charged, the protein switches into an open state in which strands ß1 and ß2 separate from the ß-barrel; the remaining structure is unaffected. In the open state the hydrophobic interior of the protein is exposed to bulk water, allowing Glu-23 to become hydrated. This illustrates several key aspects of protein electrostatics: (1) The apparent pKa of an internal ionizable group can reflect the average of the very different pKa values (open ≈4.5, closed ≫7.5) sampled in the different conformational states. (2) The high apparent dielectric constant reported by the pKa value of internal ionizable group reflects conformational reorganization. (3) The apparent pKa of internal groups can be governed by large conformational changes. (4) A single charge buried in the hydrophobic interior of a protein is sufficient to convert what might have been a transient, partially unfolded state into the dominant state in solution. This suggests a general strategy for examining inaccessible regions of the folding landscape and for engineering conformational switches driven by small changes in pH. These data also constitute a benchmark for stringent testing of the ability of computational algorithms to predict pKa values of internal residues and to reproduce pH-driven conformational transitions of proteins.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Nuclease do Micrococo/química , Conformação Proteica , Estrutura Secundária de Proteína , Cristalização , Cristalografia por Raios X , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Eletricidade Estática , Termodinâmica , Valina/química , Valina/genética , Valina/metabolismo , Água/química
2.
Biochemistry ; 56(40): 5338-5346, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28952715

RESUMO

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.


Assuntos
Nuclease do Micrococo/química , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Conformação Proteica , Termodinâmica
3.
Proc Natl Acad Sci U S A ; 111(32): 11685-90, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25074910

RESUMO

An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pKa values near 7. When buried together their pKa values appear to be normal. The ionizable moieties of the buried Glu-Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu-Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu-Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.


Assuntos
Proteínas/química , Substituição de Aminoácidos , Fenômenos Biofísicos , Cristalografia por Raios X , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Íons/química , Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eletricidade Estática , Termodinâmica
4.
Proc Natl Acad Sci U S A ; 109(18): 6945-50, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22496593

RESUMO

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.


Assuntos
Desnaturação Proteica , Dobramento de Proteína , Resposta a Proteínas não Dobradas/fisiologia , Substituição de Aminoácidos , Fenômenos Biofísicos , Cristalografia por Raios X , Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Pressão , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Solventes , Espectrometria de Fluorescência , Triptofano/química , Água/química
5.
Proteins ; 82(3): 528-34, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23999883

RESUMO

The X-ray structures of the hemoglobin from Synechococcus sp. PCC 7002 (GlbN) were solved in the ferric bis-histidine (1.44 Å resolution) and cyanide-bound (2.25 Å resolution) states with covalently attached heme. The two structures illustrate the conformational changes and cavity opening caused by exogenous ligand binding. They also reveal an unusually distorted heme, ruffled as in c cytochromes. Comparison to the solution structure demonstrates the influence of crystal packing on several structural elements, whereas comparison to GlbN from Synechocystis sp. PCC 6803 shows subtle differences in heme geometries and environment. The new structures will be instrumental in elucidating GlbN reactivity.


Assuntos
Cristalografia por Raios X/métodos , Heme/química , Hemoglobinas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Synechococcus/química , Modelos Moleculares , Conformação Proteica
6.
Proc Natl Acad Sci U S A ; 108(47): 18954-9, 2011 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-22080604

RESUMO

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 5.7 pH 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 pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. 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.


Assuntos
Arginina/química , Nuclease do Micrococo/química , Modelos Moleculares , Conformação Proteica , Guanidina/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Eletricidade Estática , Termodinâmica , Água/química
7.
J Inorg Biochem ; 259: 112654, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38959524

RESUMO

In our continued investigations of microbial globins, we solved the structure of a truncated hemoglobin from Shewanella benthica, an obligate psychropiezophilic bacterium. The distal side of the heme active site is lined mostly with hydrophobic residues, with the exception of a tyrosine, Tyr34 (CD1) and a histidine, His24 (B13). We found that purified SbHbN, when crystallized in the ferric form with polyethylene glycol as precipitant, turned into a green color over weeks. The electron density obtained from the green crystals accommodated a trans heme d, a chlorin-type derivative featuring a γ-spirolactone and a vicinal hydroxyl group on a pyrroline ring. In solution, exposure of the protein to one equivalent of hydrogen peroxide resulted in a similar green color change, but caused by the formation of multiple products. These were oxidation species released on protein denaturation, likely including heme d, and a species with heme covalently attached to the polypeptide. The Tyr34Phe replacement prevented the formation of both heme d and the covalent linkage. The ready modification of heme b by SbHbN expands the range of chemistries supported by the globin fold and offers a route to a novel heme cofactor.


Assuntos
Heme , Shewanella , Shewanella/metabolismo , Shewanella/química , Heme/química , Heme/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Cristalografia por Raios X , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/metabolismo
8.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 67(Pt 11): 1310-5, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22102223

RESUMO

Fis1 mediates mitochondrial and peroxisomal fission. It is tail-anchored to these organelles by a transmembrane domain, exposing a soluble cytoplasmic domain. Previous studies suggested that Fis1 is autoinhibited by its N-terminal region. Here, a 1.75 Å resolution crystal structure of the Fis1 cytoplasmic domain from Saccharomyces cerevisiae is reported which adopts a tetratricopeptide-repeat fold. It is observed that this fold creates a concave surface important for fission, but is sterically occluded by its N-terminal region. Thus, this structure provides a physical basis for autoinhibition and allows a detailed examination of the interactions that stabilize the inhibited state of this molecule.


Assuntos
Proteínas Mitocondriais/química , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cristalografia por Raios X , Modelos Moleculares , Homologia Estrutural de Proteína
9.
J Inorg Biochem ; 219: 111437, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33892380

RESUMO

THB1 is a monomeric truncated hemoglobin (TrHb) found in the cytoplasm of the green alga Chlamydomonas reinhardtii. The canonical heme coordination scheme in hemoglobins is a proximal histidine ligand and an open distal site. In THB1, the latter site is occupied by Lys53, which is likely to facilitate Fe(II)/Fe(III) redox cycling but hinders dioxygen binding, two features inherent to the NO dioxygenase activity of the protein. TrHb surveys show that a lysine at a position aligning with Lys53 is an insufficient determinant of coordination, and in this study, we sought to identify factors controlling lysine affinity for the heme iron. We solved the "Lys-off" X-ray structure of THB1, represented by the cyanide adduct of the Fe(III) protein, and hypothesized that interactions that differ between the known "Lys-on" structure and the Lys-off structure participate in the control of Lys53 affinity for the heme iron. We applied an experimental approach (site-directed mutagenesis, heme modification, pH titrations in the Fe(III) and Fe(II) states) and a computational approach (MD simulations in the Fe(II) state) to assess the role of heme propionate-protein interactions, distal helix capping, and the composition of the distal pocket. All THB1 modifications resulted in a weakening of lysine affinity and affected the coupling between Lys53 proton binding and heme redox potential. The results supported the importance of specific heme peripheral interactions for the pH stability of iron coordination and the ability of the protein to undergo redox reactions.


Assuntos
Heme/química , Ferro/química , Lisina/química , Hemoglobinas Truncadas/química , Chlamydomonas reinhardtii , Cristalografia por Raios X/métodos , Compostos Férricos/química , Hemoglobinas/química , Histidina/química , Concentração de Íons de Hidrogênio , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Oxirredução , Oxigenases/metabolismo , Conformação Proteica
10.
Proteins ; 77(3): 570-88, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19533744

RESUMO

Prior computational studies of the acid-unfolding behavior of staphylococcal nuclease (SNase) suggest that the pK(a) values of its carboxylic groups are difficult to reproduce with electrostatics calculations with continuum methods. To examine the molecular determinants of the pK(a) values of carboxylic groups in SNase, the pK(a) values of all 20 Asp and Glu residues were measured with multidimensional and multinuclear NMR spectroscopy in an acid insensitive variant of SNase. The crystal structure of the protein was obtained to describe the microenvironments of the carboxylic groups. Fourteen Asp and Glu residues titrate with relatively normal pK(a) values that are depressed by less than 1.1 units relative to the normal pK(a) of Asp and Glu in water. Only six residues have pK(a) values shifted by more than 1.5 units. Asp-21 has an unusually high pK(a) of 6.5, which is probably the result of interactions with other carboxylic groups at the active site. The most perturbed pK(a) values appear to be governed by hydrogen bonding and not by Coulomb interactions. The pK(a) values calculated with standard continuum electrostatics methods applied to static structures are more depressed than the measured values because Coulomb effects are exaggerated in the calculations. The problems persist even when the protein is treated with the dielectric constant of water. This can be interpreted to imply that structural relaxation is an important determinant of the pK(a) values; however, no major pH-sensitive conformational reorganization of the backbone was detected using NMR spectroscopy.


Assuntos
Ácido Aspártico/química , Ácido Glutâmico/química , Nuclease do Micrococo/química , Calibragem , Cristalografia por Raios X/métodos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática
11.
Protein Sci ; 28(1): 68-78, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30052294

RESUMO

The redox chemistry of flavoproteins is often gated by substrate and iodotyrosine deiodinase (IYD) has the additional ability to switch between reaction modes based on the substrate. Association of fluorotyrosine (F-Tyr), an inert substrate analog, stabilizes single electron transfer reactions of IYD that are not observed in the absence of this ligand. The co-crystal of F-Tyr and a T239A variant of human IYD have now been characterized to provide a structural basis for control of its flavin reactivity. Coordination of F-Tyr in the active site of this IYD closely mimics that of iodotyrosine and only minor perturbations are observed after replacement of an active site Thr with Ala. However, loss of the side chain hydroxyl group removes a key hydrogen bond from flavin and suppresses the formation of its semiquinone intermediate. Even substitution of Thr with Ser decreases the midpoint potential of human IYD between its oxidized and semiquinone forms of flavin by almost 80 mV. This decrease does not adversely affect the kinetics of reductive dehalogenation although an analogous Ala variant exhibits a 6.7-fold decrease in its kcat /Km . Active site ligands lacking the zwitterion of halotyrosine are not able to induce closure of the active site lid that is necessary for promoting single electron transfer and dehalogenation. Under these conditions, a basal two-electron process dominates catalysis as indicated by preferential reduction of nitrophenol rather than deiodination of iodophenol.


Assuntos
Dinitrocresóis/química , Iodeto Peroxidase/química , Substituição de Aminoácidos , Domínio Catalítico , Humanos , Iodeto Peroxidase/genética , Cinética , Mutação de Sentido Incorreto , Oxirredução
12.
Biophys J ; 94(8): 3208-16, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18178652

RESUMO

Although internal water molecules are essential for the structure and function of many proteins, the structural and physical factors that govern internal hydration are poorly understood. We have examined the molecular determinants of internal hydration systematically, by solving the crystal structures of variants of staphylococcal nuclease with Gln-66, Asn-66, and Tyr-66 at cryo (100 K) and room (298 K) temperatures, and comparing them with existing cryo and room temperature structures of variants with Glu-66, Asp-66, Lys-66, Glu-92 or Lys-92 obtained under conditions of pH where the internal ionizable groups are in the neutral state. At cryogenic temperatures the polar moieties of all these internal side chains are hydrated except in the cases of Lys-66 and Lys-92. At room temperature the internal water molecules were observed only in variants with Glu-66 and Tyr-66; water molecules in the other variants are probably present but they are disordered and therefore undetectable crystallographically. Each internal water molecule establishes between 3 and 5 hydrogen bonds with the protein or with other internal water molecules. The strength of interactions between internal polar side chains and water molecules seems to decrease from carboxylic acids to amides to amines. Low temperature, low cavity volume, and the presence of oxygen atoms in the cavity increase the positional stability of internal water molecules. This set of structures and the physical insight they contribute into internal hydration will be useful for the development and benchmarking of computational methods for artificial hydration of pockets, cavities, and active sites in proteins.


Assuntos
Cristalografia/métodos , Modelos Químicos , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Água/química , Simulação por Computador , Conformação Molecular , Porosidade
13.
J Phys Chem B ; 122(9): 2516-2524, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29466010

RESUMO

Thirty years ago, Hwang and Warshel suggested that a microenvironment preorganized to stabilize an ion pair would be incapable of reorganizing to stabilize the reverse ion pair. The implications were that (1) proteins have a limited capacity to reorganize, even under the influence of strong interactions, such as those present when ionizable groups are buried in the hydrophobic interior of a protein, and (2) the inability of proteins to tolerate the reversal of buried ion pairs demonstrates the limitations inherent to continuum electrostatic models of proteins. Previously we showed that when buried individually in the interior of staphylococcal nuclease, Glu23 and Lys36 have p Ka values near pH 7, but when buried simultaneously, they establish a strong interaction of ∼5 kcal/mol and have p Ka values shifted toward more normal values. Here, using equilibrium thermodynamic measurements, crystal structures, and NMR spectroscopy experiments, we show that although the reversed, individual substitutions-Lys23 and Glu36-also have p Ka values near 7, when buried together, they neither establish a strong interaction nor promote reorganization of their microenvironment. These experiments both confirm Warshel's original hypothesis and expand it by showing that it applies to reorganization, as demonstrated by our artificial ion pairs, as well as to preorganization as is commonly argued for motifs that stabilize naturally occurring ion pairs in polar microenvironments. These data constitute a challenging benchmark useful to test the ability of structure-based algorithms to reproduce the compensation between self-energy, Coulomb and polar interactions in hydrophobic environments of proteins.


Assuntos
Glutamina/química , Lisina/química , Proteínas/química , Termodinâmica , Interações Hidrofóbicas e Hidrofílicas , Íons/química , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Eletricidade Estática
14.
Biochim Biophys Acta Gen Subj ; 1862(12): 2660-2673, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30251657

RESUMO

BACKGROUND: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. METHODS: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. RESULTS: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment. CONCLUSIONS: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. GENERAL SIGNIFICANCE: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Heme/metabolismo , Lisina/metabolismo , Hemoglobinas Truncadas/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Ligantes , Mutagênese Sítio-Dirigida , Óxido Nítrico/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Conformação Proteica , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genética
15.
J Phys Chem Lett ; 9(2): 383-387, 2018 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-29266956

RESUMO

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.


Assuntos
Desoxirribonucleases/química , Espectroscopia de Ressonância Magnética , Nuclease do Micrococo/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Termodinâmica
16.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 6): 718-25, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26057801

RESUMO

THB1 is one of several group 1 truncated hemoglobins (TrHb1s) encoded in the genome of the unicellular green alga Chlamydomonas reinhardtii. THB1 expression is under the control of NIT2, the master regulator of nitrate assimilation, which also controls the expression of the only nitrate reductase in the cell, NIT1. In vitro and physiological evidence suggests that THB1 converts the nitric oxide generated by NIT1 into nitrate. To aid in the elucidation of the function and mechanism of THB1, the structure of the protein was solved in the ferric state. THB1 resembles other TrHb1s, but also exhibits distinct features associated with the coordination of the heme iron by a histidine (proximal) and a lysine (distal). The new structure illustrates the versatility of the TrHb1 fold, suggests factors that stabilize the axial ligation of a lysine, and highlights the difficulty of predicting the identity of the distal ligand, if any, in this group of proteins.


Assuntos
Proteínas de Algas/química , Chlamydomonas reinhardtii/química , Heme/química , Histidina/química , Lisina/química , Nitrato Redutase/química , Hemoglobinas Truncadas/química , Proteínas de Algas/genética , Motivos de Aminoácidos , Chlamydomonas reinhardtii/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ferro/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Nitrato Redutase/genética , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia Estrutural de Proteína , Hemoglobinas Truncadas/genética
17.
Structure ; 20(6): 1071-85, 2012 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-22632835

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Nuclease do Micrococo/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Estabilidade Enzimática , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Nuclease do Micrococo/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Desdobramento de Proteína , Staphylococcus aureus/enzimologia , Termodinâmica
18.
J Mol Biol ; 389(1): 34-47, 2009 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-19324049

RESUMO

The pK(a) values of internal ionizable groups are usually very different from the normal pK(a) values of ionizable groups in water. To examine the molecular determinants of pK(a) values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pK(a), whereas Asp38 and Glu38 titrate with elevated pK(a) values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pK(a) of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pK(a) values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pK(a) values of internal groups in proteins. This study suggests that improvements to computational methods for pK(a) calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.


Assuntos
Aminoácidos Acídicos/química , Aminoácidos Básicos/química , Nuclease do Micrococo/química , Substituição de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/química , Estrutura Secundária de Proteína , Análise Espectral , Propriedades de Superfície , Titulometria
19.
Protein Sci ; 17(5): 833-45, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18369193

RESUMO

Previously we reported that Lys, Asp, and Glu residues at positions 66 and 92 in staphylococcal nuclease (SNase) titrate with pK(a) values shifted by up to 5 pK(a) units in the direction that promotes the neutral state. In contrast, the internal Lys-38 in SNase titrates with a normal pK(a). The crystal structure of the L38K variant shows that the side chain of Lys-38 is buried. The ionizable moiety is approximately 7 A from solvent and ion paired with Glu-122. This suggests that the pK(a) value of Lys-38 is normal because the energetic penalty for dehydration is offset by a favorable Coulomb interaction. However, the pK(a) of Lys-38 was also normal when Glu-122 was replaced with Gln or with Ala. Continuum electrostatics calculations were unable to reproduce the pK(a) of Lys-38 unless the protein was treated with an artificially high dielectric constant, consistent with structural reorganization being responsible for the normal pK(a) value of Lys-38. This reorganization must be local because circular dichroism and NMR spectroscopy indicate that the L38K protein is native-like under all conditions studied. In molecular dynamics simulations, the ion pair between Lys-38 and Glu-122 is unstable. The simulations show that a minor rearrangement of a loop is sufficient to allow penetration of water to the amino moiety of Lys-38. This illustrates both the important roles of local flexibility and water penetration as determinants of pK(a) values of ionizable groups buried near the protein-water interface, and the challenges faced by structure-based pK(a) calculations in reproducing these effects.


Assuntos
Lisina/química , Nuclease do Micrococo/química , Água/química , Substituição de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Leucina/química , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Eletricidade Estática , Titulometria
20.
J Mol Biol ; 379(5): 1045-62, 2008 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-18499123

RESUMO

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK(a) values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK(a) values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK(a) values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK(a) was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N(epsilon2) state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK(a) calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK(a) values are governed by short-range polar and Coulomb interactions.


Assuntos
Nuclease do Micrococo/química , Substituição de Aminoácidos , Cristalografia por Raios X , Estabilidade Enzimática , Histidina/química , Concentração de Íons de Hidrogênio , Nuclease do Micrococo/genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Salinidade , Eletricidade Estática , Termodinâmica
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