Comparison of Proteolytic Susceptibility in Phosphoglycerate Kinases from Yeast and E. coli: Modulation of Conformational Ensembles Without Altering Structure or Stability

Young, Tracy A.; Skordalakes, Emmanuel; Marqusee, Susan

doi:10.1016/j.jmb.2007.02.077

Cited by 40 publications

(48 citation statements)

References 35 publications

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“…The exposure of the R127/T128 cleavage site is not a rare process, with equilibrium values ranging between 5×10 -4 and 0.0124. Similar studies of other proteins have found values more than 200-fold lower (50,61,66). It may also be possible that the magnitude of motion required for transition between the folded and protease-accessible states is far greater than that required for biological activity in the native particles, resulting in effectively higher exposure for native interactions.…”

Section: Discussionmentioning

confidence: 76%

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

Hilmer

Zlotnick

Bothner

2008

Journal of Molecular Biology

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Functional analysis of Hepatitis B virus (HBV) core particles has associated a number of biological roles with the C-terminus of the capsid protein. One set of functions require the C-terminus to be on the exterior of the capsid, while others place this domain on the interior. According to the crystal structure of the capsid, this segment is strictly internal to the capsid shell and buried at a proteinprotein interface. Using kinetic hydrolysis, a form of protease digestion assayed by SDS-PAGE and mass spectrometry, the structurally and biologically important C-terminal region of HBV capsid protein assembly domain (Cp149, residues 1-149) has been shown to be dynamic in both dimer and capsid forms. HBV is an enveloped virus with a T=4 icosahedral core that is composed of 120 copies of a homodimer capsid protein. Free dimer and assembled capsid forms of the protein are readily hydrolyzed by trypsin and thermolysin, around residues 127-128, indicating that this region is dynamic and exposed to the capsid surface. The measured conformational equilibria have an opposite temperature dependence between free dimer and assembled capsid. This work helps to explain the previously described allosteric regulation of assembly and functional properties of a buried domain. These observations make a critical connection between structure, dynamics, and function: made possible by the first quantitative measurements of conformational equilibria and rates of conversion between protein conformers for a megadalton complex.

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Section: Discussionmentioning

confidence: 76%

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

Hilmer

Zlotnick

Bothner

2008

Journal of Molecular Biology

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“…This data is particularly relevant since a number of reports on stability changes do not translate into changes in proteolytic susceptibility. 40,41 The dependence of protease susceptibility on stability was mainly reported from the studies on homologous proteins from extremophiles. 2,6 Comparison of crystal structures of several homologous proteins from extremophiles have shown that the crystallographic B-factors are negatively correlated with the thermophilicity of proteins.…”

Section: Discussionmentioning

confidence: 99%

“…44 Similarly, enormous differences in proteolytic susceptibilities of yeast and E. coli phosphoglycerate kinase has been reported which posses very similar three-dimensional structure as well as global stability. 41 A set of mutants generated in the N-terminal domain of k repressor showed that thermostable mutants are less susceptible to proteolytic attack. 4 Similarly, mutants of ribonuclease barnase from Bacillus amyloliquefaciens evolved for stability displayed great improvement in their proteolytic resistance with little improvement in their thermodynamic stabilities, suggesting factors governing proteolytic resistance might be different from those governing thermodynamic stability.…”

Section: Discussionmentioning

confidence: 99%

Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

et al. 2012

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Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of~16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm app ) by 1-6°C resulting in an increase in free energy of unfolding (DG unf ) by 0.04-1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with DG unf , (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (DDG unf 5 1.57 kcal 21 ) justifies the robustness of this relation.

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“…A failure of R106W either to fold properly (39) or to undergo the normal conformational changes upon substrate binding is a possibility under investigation. Single amino acid substitutions tend to result in altered dynamics and energetics rather than an overall change in three-dimensional structure (40), and examining these properties is an important future goal. Understanding the different defects in MeCP2 mutations that lead to reduced interactions with DNA and chromatin as reported here will provide information critical to the identification of small molecules that may restore function and lead to effective mutation-targeted RTT therapies.…”

Section: Discussionmentioning

confidence: 99%

MeCP2-Chromatin Interactions Include the Formation of Chromatosome-like Structures and Are Altered in Mutations Causing Rett Syndrome

Nikitina

Ghosh

Horowitz-Scherer

et al. 2007

Journal of Biological Chemistry

103

112

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Sporadic mutations in hMeCP2 (human methylated DNAbinding protein 2) cause the majority of cases of the X-linked neurodevelopmental disease known as Rett syndrome (RTT), 2 a severe autism spectrum disorder (reviewed in Refs. 1-3). The disease results in a diverse range of debilitating physical and neurological symptoms that typically make their initial appearance in the first 6 -18 months of life in affected girls. Although most RTT cases result from a loss of function effect, mutations that increase the MeCP2 dose may also give rise to similar symptoms, a finding seen also in mice engineered to synthesize additional MeCP2 (4). Extensive research has suggested that MeCP2 acts as a transmitter of epigenetic information by binding to symmetrically methylated CpG dinucleotides, recruiting complexes that include histone deacetylase and methyltransferase, and leading to local transcriptional repression. However, there is also considerable evidence that MeCP2 governs additional processes such as chromatin condensation (5, 6) and that its function is not restricted to transcriptional repression (3).To better understand MeCP2 function, we are studying the basic interactions between MeCP2 and its DNA and chromatin substrates. In addition to a methylation-dependent interaction with DNA that is mediated by the methylated DNA-binding domain (MBD), MeCP2 has a C-terminal chromatin-binding region as well as additional DNA-binding regions (5, 6). MeCP2 binding contributes to the formation in nucleosomal arrays of a distinctive structural motif in which the entering and exiting linker DNA segments are brought into close juxtaposition forming a "stem." The stem motif appears very similar to structures induced by histone H1 on mono-nucleosomes and polynucleosomes (7,8) and is thought to initiate the zigzag conformation and compaction of H1-containing chromatin. MeCP2 shares with H1 the ability to induce chromatin compaction, but the multiple chromatin-binding regions of MeCP2 lead to a higher level of condensation (9).A distinctive property of H1-containing chromatin is the protection from micrococcal nuclease (Mnase) digestion of ϳ20 bp of DNA beyond the ϳ146 bp of the nucleosome core particle (NCP). There is considerable evidence that the globular domain of H1 binds near the linker entry-exit site of the nucleosome (reviewed in Ref. 10). However, the location of the ϳ20 bp of protected DNA with respect to the parent nucleosome is influenced by the underlying DNA sequence (11), and the detailed molecular structure of the H1-containing unit, termed the chromatosome (12), remains controversial (10).In the present study we show that hMeCP2, like H1, provides specific protection of linker DNA. However, the two proteins differ significantly in the length of protected DNA. With the

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Comparison of Proteolytic Susceptibility in Phosphoglycerate Kinases from Yeast and E. coli: Modulation of Conformational Ensembles Without Altering Structure or Stability

Cited by 40 publications

References 35 publications

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

MeCP2-Chromatin Interactions Include the Formation of Chromatosome-like Structures and Are Altered in Mutations Causing Rett Syndrome

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