Hydrogen Bonding Markedly Reduces the pK of Buried Carboxyl Groups in Proteins

Thurlkill, Richard L.; Grimsley, Gerald R.; Scholtz, J. Martin; Pace, C. Nick

doi:10.1016/j.jmb.2006.07.056

Cited by 64 publications

(67 citation statements)

References 46 publications

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“…Consistent with this observation, we identified 30 charged or polar residues including Glu 1829 (12) buried at the A2-interactive surface that may potentially form inter-domain hydrogen bonds, based upon spatial separations of Ͻ2.8 Å, contributing to the overall binding energy (13,14). To determine a role for these residues in the stability of factor VIIIa, as well as the factor VIII procofactor, we employed a site-directed mutagenesis approach where each of these residues was individually replaced by Ala (Phe for Tyr residues) and the resulting factor VIII variants were stably expressed as B-domainless factor VIII in baby hamster kidney cells.…”

supporting

confidence: 75%

Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

Wakabayashi

Fay

2008

Journal of Biological Chemistry

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Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8 Å were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55°C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.Factor VIII, a plasma protein that participates in the blood coagulation cascade, is decreased or defective in individuals with hemophilia A. Factor VIII circulates as a non-covalent, metal ion-dependent heterodimer consisting of a heavy chain comprised of A1(a1)A2(a2)B 2 domains and a light chain comprised of (a3)A3C1C2 domains (see Ref. 1 for review). The factor VIII procofactor is activated by limited proteolysis catalyzed by thrombin or factor Xa to yield the cofactor, factor VIIIa, following cleavages at the a1A2, a2B, and a3A3 junctions. Thus, factor VIIIa is a heterotrimer of subunits designated A1, A2, and A3C1C2. Factor VIIIa functions as a cofactor for the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa (see Ref. 1 for review). The role of the cofactor in the intrinsic factor Xase complex is to increase the catalytic efficiency of this reaction by several orders of magnitude. Factor Xase is self-dampening as a result of instability in the factor VIIIa cofactor due to weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer (2, 3). Limited information is available regarding the association of the A2 subunit in factor VIIIa. Fluorescence energy transfer results (4) and activity assays following swapping the human A1 domain for the porcine homolog (5) suggest that interactions o...

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supporting

confidence: 75%

Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

Wakabayashi

Fay

2008

Journal of Biological Chemistry

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“…Our titrations reveal the additive nature of the effect of zeaxanthin and PsbS on the pK a of protonable residues associated with qE. The apparent pK a of amino acids has been shown to strongly depend upon their environment (55)(56)(57). Hydrogen bonding, steric hindrance, and the di-electric constant of the environment can all affect the pK a of amino acids (55)(56)(57).…”

Section: Methodsmentioning

confidence: 71%

“…The apparent pK a of amino acids has been shown to strongly depend upon their environment (55)(56)(57). Hydrogen bonding, steric hindrance, and the di-electric constant of the environment can all affect the pK a of amino acids (55)(56)(57). For instance the pK a of the carboxyl group on aspartate can be as low as 2.4 when exposed to water due to hydrogen bonding, whereas in hydrophobic environments the pK a can be as high as 6.4 (56).…”

Section: Methodsmentioning

confidence: 99%

Restoration of Rapidly Reversible Photoprotective Energy Dissipation in the Absence of PsbS Protein by Enhanced ΔpH

Johnson

Ruban

2011

Journal of Biological Chemistry

122

105

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Variations in the light environment require higher plants to regulate the light harvesting process. Under high light a mechanism known as non-photochemical quenching (NPQ) is triggered to dissipate excess absorbed light energy within the photosystem II (PSII) antenna as heat, preventing photodamage to the reaction center. The major component of NPQ, known as qE, is rapidly reversible in the dark and dependent upon the transmembrane proton gradient (⌬pH), formed as a result of photosynthetic electron transport. Using diaminodurene and phenazine metasulfate, mediators of cyclic electron flow around photosystem I, to enhance ⌬pH, it is demonstrated that rapidly reversible qE-type quenching can be observed in intact chloroplasts from Arabidopsis plants lacking the PsbS protein, previously believed to be indispensible for the process. The qE in chloroplasts lacking PsbS significantly quenched the level of fluorescence when all PSII reaction centers were in the open state (F o state), protected PSII reaction centers from photoinhibition, was modulated by zeaxanthin and was accompanied by the qEtypical absorption spectral changes, known as ⌬A 535 . Titrations of the ⌬pH dependence of qE in the absence of PsbS reveal that this protein affects the cooperativity and sensitivity of the photoprotective process to protons. The roles of PsbS and zeaxanthin are discussed in light of their involvement in the control of the proton-antenna association constant, pK, via regulation of the interconnected phenomena of PSII antenna reorganization/aggregation and hydrophobicity.Sunlight can fluctuate frequently and dramatically in both intensity and spectral quality during the day. The photosynthetic membrane of higher plants has evolved to operate efficiently under such light conditions. In shade, large arrays of antenna complexes efficiently harvest and deliver photon energy to the photochemical reaction centers for use in photosynthesis. In high light the membrane switches to a photoprotective state where the excess absorbed energy, which has the potential to cause damage, is safely dissipated as heat (1, 2). The amount of energy dissipation can be monitored by the nonphotochemical quenching of chlorophyll fluorescence (NPQ) 2 (1, 2). The major component of NPQ, qE, is rapidly reversible in the dark and is dependent upon the level of the transmembrane proton gradient (⌬pH). ⌬pH acts as a feedback signal indicating the degree of saturation of photosynthetic electron transport (1, 2). ⌬pH can be generated by linear electron flow (LEF), which involves the oxidation of water by photosystem II (PSII) and the transfer of electrons to NADP ϩ via the cytochrome b 6 f and photosystem I (PSI) complexes (3). In addition, cyclic electron flow (CEF), which involves the transfer of electrons in a closed loop around cytochrome b 6 f and PSI was reported to contribute to ⌬pH (4 -6). The contribution of CEF to ⌬pH generation is essential for maintaining the correct ATP/ NADPH ratio for CO 2 fixation in the chloroplast stroma (6). Imbalance in the AT...

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“…If the scheme in Figure 1B is correct, why does PsbS make the LHCII antenna more sensitive to lumen pH? Is it because it somehow enhances hydrophobicity of the environment of proton-receiving amino acids, which would certainly make their pK values higher (Mehler et al, 2002;Thurlkill et al, 2006)? Also, while both PsbS and zeaxanthin promote rapid formation of NPQ (Li et al, 2000;Demmig-Adams et al, 1989), why has the former an acceleratory and the latter an inhibitory effect on its recovery, as well as opposite effects on chlorophyll excited state relaxation dynamics (Sylak-Glassman et al, 2014)?…”

Section: Change: Lhcii Rearrangements/aggregation and The Formation Omentioning

confidence: 99%

Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage

2016

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We review the mechanism underlying nonphotochemical chlorophyll fluorescence quenching (NPQ) and its role in protecting plants against photoinhibition. This review includes an introduction to this phenomenon, a brief history of major milestones in our understanding of NPQ, definitions, and a discussion of quantitative measurements of NPQ. We discuss the current knowledge and unknown aspects in the NPQ scenario, including the following: DpH, the proton gradient (trigger); lightharvesting complex II (LHCII), PSII light harvesting antenna (site); and changes in the antenna induced by DpH (change), which lead to the creation of the quencher. We conclude that the minimum requirements for NPQ in vivo are DpH, LHCII complexes, and the PsbS protein. We highlight the most important unknown in the NPQ scenario, the mechanism by which PsbS acts upon the LHCII antenna. Finally, we describe a novel, emerging technology for assessing the photoprotective "power" of NPQ and the important findings obtained through this technology.

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Hydrogen Bonding Markedly Reduces the pK of Buried Carboxyl Groups in Proteins

Cited by 64 publications

References 46 publications

Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

Restoration of Rapidly Reversible Photoprotective Energy Dissipation in the Absence of PsbS Protein by Enhanced ΔpH

Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage

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