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Jonathan King

Publications and source records attributed to Jonathan King.

At least 19 recordsLinked to original sources
PubMed2006

Mechanism of the highly efficient quenching of tryptophan fluorescence in human gammaD-crystallin.

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.

Amino Acid Sequence↗
PubMed2006

Glutamine deamidation destabilizes human gammaD-crystallin and lowers the kinetic barrier to unfolding.

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gammaD-crystallin (HgammaD-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HgammaD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HgammaD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.

Glutamine↗
PubMed2006

Fold recognition and accurate sequence-structure alignment of sequences directing beta-sheet proteins.

The ability to predict structure from sequence is particularly important for toxins, virulence factors, allergens, cytokines, and other proteins of public health importance. Many such functions are represented in the parallel beta-helix and beta-trefoil families. A method using pairwise beta-strand interaction probabilities coupled with evolutionary information represented by sequence profiles is developed to tackle these problems for the beta-helix and beta-trefoil folds. The algorithm BetaWrapPro employs a "wrapping" component that may capture folding processes with an initiation stage followed by processive interaction of the sequence with the already-formed motifs. BetaWrapPro outperforms all previous motif recognition programs for these folds, recognizing the beta-helix with 100% sensitivity and 99.7% specificity and the beta-trefoil with 100% sensitivity and 92.5% specificity, in crossvalidation on a database of all nonredundant known positive and negative examples of these fold classes in the PDB. It additionally aligns 88% of residues for the beta-helices and 86% for the beta-trefoils accurately (within four residues of the exact position) to the structural template, which is then used with the side-chain packing program SCWRL to produce 3D structure predictions. One striking result has been the prediction of an unexpected parallel beta-helix structure for a pollen allergen, and its recent confirmation through solution of its structure. A Web server running BetaWrapPro is available and outputs putative PDB-style coordinates for sequences predicted to form the target folds.

Algorithms↗
PubMed2006

Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery.

The mechanisms by which most double-stranded DNA viruses package and release their genomic DNA are not fully understood. Single particle cryo-electron microscopy and asymmetric 3D reconstruction reveal the organization of the complete bacteriophage P22 virion, including the protein channel through which DNA is first packaged and later ejected. This channel is formed by a dodecamer of portal proteins and sealed by a tail hub consisting of two stacked barrels capped by a protein needle. Six trimeric tailspikes attached around this tail hub are kinked, suggesting a functional hinge that may be used to trigger DNA release. Inside the capsid, the portal's central channel is plugged by densities interpreted as pilot/injection proteins. A short rod-like density near these proteins may be the terminal segment of the dsDNA genome. The coaxially packed DNA genome is encapsidated by the icosahedral shell. This complete structure unifies various biochemical, genetic, and crystallographic data of its components from the past several decades.

Bacteriophage P22↗
PubMed2006

An elongated spine of buried core residues necessary for in vivo folding of the parallel beta-helix of P22 tailspike adhesin.

The parallel beta-helix is an elongated beta-sheet protein domain associated with microbial virulence factors, toxins, viral adhesins, and allergens. Long stacks of similar, buried residues are a prominent feature of this fold, as well as the polypeptide chain fold of an amyloid structure. The 13-rung, right-handed, parallel beta-helix of the homotrimeric P22 tailspike adhesin exhibits predominantly hydrophobic stacks. The role of these stacked residues in the folding and stabilization of the protein is unclear. Through scanning alanine mutagenesis we have identified a folding spine of stacked residues in continuous contact along the length of P22 tailspike's beta-helix domain that is necessary for folding within cells. Nearly all chains carrying alanine substitutions of the 103 buried nonalanines were defective in folding in vivo at 37 degrees C. However, the majority of these chains successfully reached a native state, stable to >80 degrees C, when folded inside cells at low temperatures. Thus, nearly the entire buried core was critical for in vivo beta-helix folding but negligible for stability. Folding at 18 degrees C revealed the minimal folding spine of 29 nonglycine stack positions that were intolerant to alanine substitution. These results indicate that a processive folding mechanism, dependent on stacking contacts, controls beta-helix formation. Such a stepwise folding pathway offers a new target for drug design against this class of microbial virulence factors.

Amino Acid Substitution↗
PubMed2006

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus.

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with approximately 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.

Bacteriophages↗
PubMed2006

Frequencies of hydrophobic and hydrophilic runs and alternations in proteins of known structure.

Patterns of alternation of hydrophobic and polar residues are a profound aspect of amino acid sequences, but a feature not easily interpreted for soluble proteins. Here we report statistics of hydrophobicity patterns in proteins of known structure in a current protein database as compared with results from earlier, more limited structure sets. Previous studies indicated that long hydrophobic runs, common in membrane proteins, are underrepresented in soluble proteins. Long runs of hydrophobic residues remain significantly underrepresented in soluble proteins, with none longer than 16 residues observed. These long runs most commonly occur as buried alpha helices, with extended hydrophobic strands less common. Avoiding aggregation of partially folded intermediates during intracellular folding remains a viable explanation for the rarity of long hydrophobic runs in soluble proteins. Comparison between database editions reveals robustness of statistics on aqueous proteins despite an approximately twofold increase in nonredundant sequences. The expanded database does now allow us to explain several deviations of hydrophobicity statistics from models of random sequence in terms of requirements of specific secondary structure elements. Comparison to prior membrane-bound protein sequences, however, shows significant qualitative changes, with the average hydrophobicity and frequency of long runs of hydrophobic residues noticeably increasing between the database editions. These results suggest that the aqueous proteins of solved structure may represent an essentially complete sample of the universe of aqueous sequences, while the membrane proteins of known structure are not yet representative of the universe of membrane-associated proteins, even by relatively simple measures of hydrophobic patterns.

Computational Biology↗
PubMed2005

Stalled folding mutants in the triple beta-helix domain of the phage P22 tailspike adhesin.

The trimeric bacteriophage P22 tailspike adhesin exhibits a domain in which three extended strands intertwine, forming a single turn of a triple beta-helix. This domain contains a single hydrophobic core composed of residues contributed by each of the three sister polypeptide chains. The triple beta-helix functions as a molecular clamp, increasing the stability of this elongated structural protein. During folding of the tailspike protein, the last precursor before the native state is a partially folded trimeric intermediate called the protrimer. The transition from the protrimer to the native state results in a structure that is resistant to denaturation by heat, chemical denaturants, and proteases. Random mutations were made in the region encoding residues 540-548, where the sister chains begin to wrap around each other. From a set of 26 unique single amino acid substitutions, we characterized mutations at G546, N547, and I548 that retarded or blocked the protrimer to native trimer transition. In contrast, many non-conservative substitutions were tolerated at residues 540-544. Sucrose gradient analysis showed that protrimer-like mutants had reduced sedimentation, 8.0 S to 8.3 S versus 9.3 S for the native trimer. Mutants affected in the protrimer to native trimer transition were also destabilized in their native state. These data suggest that the folding of the triple beta-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.

Adhesins, Bacterial↗
PubMed2005

Monoclonal antibody epitope mapping describes tailspike beta-helix folding and aggregation intermediates.

There is growing interest in understanding how the cellular environment affects protein folding mechanisms, but most spectroscopic methods for monitoring folding in vitro are unsuitable for experiments in vivo or in other complex mixtures. Monoclonal antibody binding represents a sensitive structural probe that can be detected against the background of other cellular components. A panel of antibodies has been raised against Salmonella typhimurium phage P22 tailspike. In this report, nine alpha-tailspike antibody binding epitopes were characterized by measuring the binding of these monoclonal antibodies to tailspike variants bearing surface point mutations. These results reveal that the antibody epitopes are distributed throughout the tailspike structure, with several clustered in the central parallel beta-helix domain. The ability of each antibody to distinguish between tailspike conformational states was assessed by measuring antibody binding to tailspike in vitro refolding intermediates. Interestingly, the binding of all but one of the nine antibodies is sensitive to the tailspike conformational state. Whereas several antibodies bind preferentially to the tailspike native structure, the structural features that comprise the binding epitopes form with different rates. In addition, two antibodies preferentially recognize early refolding intermediates. Combined with the epitope mapping, these results indicate portions of the beta-helix form early during refolding, perhaps serving as a scaffold for the formation of additional structure. Finally, three of the antibodies show enhanced binding to non-native, potentially aggregation-prone tailspike conformations. The refolding results indicate these non-native conformations form early during the refolding reaction, long before the appearance of native tailspike.

Antibodies, Monoclonal↗
PubMed2005

Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin.

Human gammaD-crystallin (HgammaD-Crys) is a monomeric eye lens protein composed of two highly homologous beta-sheet domains. The domains interact through interdomain side chain contacts forming two structurally distinct regions, a central hydrophobic cluster and peripheral residues. The hydrophobic cluster contains Met43, Phe56, and Ile81 from the N-terminal domain (N-td) and Val132, Leu145, and Val170 from the C-terminal domain (C-td). Equilibrium unfolding/refolding of wild-type HgammaD-Crys in guanidine hydrochloride (GuHCl) was best fit to a three-state model with transition midpoints of 2.2 and 2.8 M GuHCl. The two transitions likely corresponded to sequential unfolding/refolding of the N-td and the C-td. Previous kinetic experiments revealed that the C-td refolds more rapidly than the N-td. We constructed alanine substitutions of the hydrophobic interface residues to analyze their roles in folding and stability. After purification from E. coli, all mutant proteins adopted a native-like structure similar to wild type. The mutants F56A, I81A, V132A, and L145A had a destabilized N-td, causing greater population of the single folded domain intermediate. Compared to wild type, these mutants also had reduced rates for productive refolding of the N-td but not the C-td. These data suggest a refolding pathway where the domain interface residues of the refolded C-td act as a nucleating center for refolding of the N-td. Specificity of domain interface interactions is likely important for preventing incorrect associations in the high protein concentrations of the lens nucleus.

Circular Dichroism↗
PubMed2005

Interdomain side-chain interactions in human gammaD crystallin influencing folding and stability.

Human gammaD crystallin (HgammaD-Crys) is a two domain, beta-sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of HgammaD-Crys are associated with juvenile-onset cataracts. Features of the interface between the two domains conserved among gamma-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In HgammaD-Crys these pairs are Gln54/Gln143 and Arg79/Met147. We previously reported contributions of the hydrophobic cluster residues to protein stability. In this study alanine substitutions of the flanking residue pairs were constructed and analyzed. Equilibrium unfolding/refolding experiments at 37 degrees C revealed a plateau in the unfolding/refolding transitions, suggesting population of a partially folded intermediate with a folded C-terminal domain (C-td) and unfolded N-terminal domain (N-td). The N-td was destabilized by substituting residues from both domains. In contrast, the C-td was not significantly affected by substitutions of either domain. Refolding rates of the N-td were significantly decreased for mutants of either domain. In contrast, refolding rates of the C-td were similar to wild type for mutants of either domain. Therefore, domain interface residues of the folded C-td probably nucleate refolding of the N-td. We suggest that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. Glutamine and methionine side chains are among the residues covalently damaged in aged and cataractous lenses. Such damage may generate partially unfolded, aggregation- prone conformations of HgammaD-Crys that could be significant in cataract.

Amino Acid Substitution↗
PubMed2005

Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state.

Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37 degrees C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1-2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.

Biophysical Phenomena↗
PubMed2004

Probing folding and fluorescence quenching in human gammaD crystallin Greek key domains using triple tryptophan mutant proteins.

Human gammaD crystallin (HgammaD-Crys), a major component of the human eye lens, is a 173-residue, primarily beta-sheet protein, associated with juvenile and mature-onset cataracts. HgammaD-Crys has four tryptophans, with two in each of the homologous Greek key domains, which are conserved throughout the gamma-crystallin family. HgammaD-Crys exhibits native-state fluorescence quenching, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may influence the lens response to ultraviolet light or the protection of the retina from ambient ultraviolet damage. To provide fluorescence reporters for each quadrant of the protein, triple mutants, each containing three tryptophan-to-phenylalanine substitutions and one native tryptophan, have been constructed and expressed. Trp 42-only and Trp 130-only exhibited fluorescence quenching between the native and denatured states typical of globular proteins, whereas Trp 68-only and Trp 156-only retained the anomalous quenching pattern of wild-type HgammaD-Crys. The three-dimensional structure of HgammaD-Crys shows Tyr/Tyr/His aromatic cages surrounding Trp 68 and Trp 156 that may be the source of the native-state quenching. During equilibrium refolding/unfolding at 37 degrees C, the tryptophan fluorescence signals indicated that domain I (W42-only and W68-only) unfolded at lower concentrations of GdnHCl than domain II (W130-only and W156-only). Kinetic analysis of both the unfolding and refolding of the triple-mutant tryptophan proteins identified an intermediate along the HgammaD-Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of HgammaD-Crys.

Amino Acid Substitution↗
PubMed2004

Buried hydrophobic side-chains essential for the folding of the parallel beta-helix domains of the P22 tailspike.

The processive beta-strands and turns of a polypeptide parallel beta-helix represent one of the topologically simplest beta-sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel beta-helix followed by an interdigitated section of triple-stranded beta-helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two beta-helix domains and extend into the core of the contiguous triple beta-prism domain. To test whether these side-chain stacks represent essential residues for driving the chain into the correct fold, each of three stacked phenylalanine residues within the buried core were substituted with less bulky amino acids. The mutant chains with alanine in place of phenylalanine were defective in intracellular folding. The chains accumulated exclusively in the aggregated inclusion body state regardless of temperature of folding. These severe folding defects indicate that the stacked phenylalanine residues are essential for correct parallel beta-helix folding. Replacement of the same phenylalanine residues with valine or leucine also impaired folding in vivo, but with less severity. Mutants were also constructed in a second buried stack that extends into the intertwined triple-stranded beta-helix and contiguous beta-prism regions of the protein. These mutants exhibited severe defects in later stages of chain folding or assembly, accumulating as misfolded but soluble multimeric species. The results indicate that the formation of the buried hydrophobic stacks is critical for the correct folding of the parallel beta-helix, triple-stranded beta-helix, and beta-prism domains in the tailspike protein.

Amino Acid Substitution↗
PubMed2004

Protein folding failure sets high-temperature limit on growth of phage P22 in Salmonella enterica serovar Typhimurium.

The high-temperature limit for growth of microorganisms differs greatly depending on their species and habitat. The importance of an organism's ability to manage thermal stress is reflected in the ubiquitous distribution of the heat shock chaperones. Although many chaperones function to reduce protein folding defects, it has been difficult to identify the specific protein folding pathways that set the high-temperature limit of growth for a given microorganism. We have investigated this for a simple system, phage P22 infection of Salmonella enterica serovar Typhimurium. Production of infectious particles exhibited a broad maximum of 150 phage per cell when host cells were grown at between 30 and 39 degrees C in minimal medium. Production of infectious phage declined sharply in the range of 40 to 41 degrees C, and at 42 degrees C, production had fallen to less than 1% of the maximum rate. The host cells maintained optimal division rates at these temperatures. The decrease in phage infectivity was steeper than the loss of physical particles, suggesting that noninfectious particles were formed at higher temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the tailspike adhesins assembled on phage particles purified from cultures incubated at higher temperatures. The infectivity of these particles was restored by in vitro incubation with soluble tailspike trimers. Examination of tailspike folding and assembly in lysates of phage-infected cells confirmed that the fraction of polypeptide chains able to reach the native state in vivo decreased with increasing temperature, indicating a thermal folding defect rather than a particle assembly defect. Thus, we believe that the folding pathway of the tailspike adhesin sets the high-temperature limit for P22 formation in Salmonella serovar Typhimurium.

Bacteriophage P22↗
PubMed2003

In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation.

Human gammaD crystallin (HgammaD-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily beta-sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HgammaD-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type HgammaD-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37 degrees C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type HgammaD-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of HgammaD-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The HgammaD-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of HgammaD-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo.

Cataract↗
PubMed2002

The reactivity and oxidation pathway of cysteine 232 in recombinant human alpha 1-antitrypsin.

Oxidative damage to the sulfur-containing amino acids, methionine and cysteine, is a major concern in biotechnology and medicine. alpha1-Antitrypsin, which is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors, contains nine methionines and a single cysteine in its primary sequence. Although it is known that methionine oxidation in the protein active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of its unpaired thiol, Cys-232. In this study, the thiol-modifying reagent NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole) was used to label peroxide-modified alpha1-antitrypsin and demonstrate that the Cys-232 in vitro oxidation pathway begins with a stable sulfenic acid intermediate and is followed by the formation of sulfinic and cysteic acid in successive steps. pH-dependent reactivity with hydrogen peroxide showed that Cys-232 has a pK(a) of 6.86 +/- 0.05, a value that is more than 1.5 pH units lower than that of a typical protein thiol. pH-induced conformational changes in the region surrounding Cys-232 were also examined and indicate that mildly acidic conditions induce a conformation that enhances Cys-232 reactivity. In summary, this work provides new insights into alpha1-antitrypsin reactivity in oxidizing environments and shows that a unique structural environment renders its unpaired thiol, Cys-232, its most reactive amino acid.

Cysteine↗