CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core

Baliga, Chetana; Selmke, Benjamin; Worobiew, Irina; Borbat, Peter P.; Sarma, Siddhartha P.; Trommer, Wolfgang E.; Varadarajan, Raghavan; Aghera, Nilesh

doi:10.1016/j.bpj.2019.01.026

Cited by 8 publications

(5 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Binding of epigallocatechin gallate with the MG state of the protein indicated sufficient extent of structural features in the intermediate state to allow binding to the incoming ligand molecules (Radibratovic et al 2019). Formation of the MG state in homodimeric CcD B [controller of cell death B] protein has been characterized spectroscopically (Baliga et al 2019). That study provided insights into structural and dynamic properties of a low-pH state of CcD B.…”

Section: Recent Qualitative Studies On the Mg Statementioning

confidence: 96%

A look back at the molten globule state of proteins: thermodynamic aspects

Judy

Kishore

2019

Biophys Rev

View full text Add to dashboard Cite

Interest in protein folding intermediates lies in their significance to protein folding pathways. The molten globule (MG) state is one such intermediate lying on the kinetic (and sometimes thermodynamic) pathway between native and unfolded states. Development of our qualitative and quantitative understanding of the MG state can provide deeper insight into the folding pathways and hence potentially facilitate solution of the protein folding problem. An extensive look at literature suggests that most studies into protein MG states have been largely qualitative. Attempts to obtain quantitative insights into MG states have involved application of high-sensitivity calorimetry (differential scanning calorimetry and isothermal titration calorimetry). This review addresses the progress made in this direction by discussing the knowledge gained to date, along with the future promise of calorimetry, in providing quantitative information on the structural features of MG states. Particular attention is paid to the question of whether such states share common structural features or not. The difference in the nature of the transition from the MG state to the unfolded state, in terms of cooperativity, has also been addressed and discussed.

show abstract

Section: Recent Qualitative Studies On the Mg Statementioning

confidence: 96%

A look back at the molten globule state of proteins: thermodynamic aspects

Judy

Kishore

2019

Biophys Rev

View full text Add to dashboard Cite

show abstract

“…Later, a DMG intermediate of barstar protein was also observed as an early unfolding intermediate during the denaturant-induced unfolding . Recently, a DMG-like intermediate was observed experimentally during the low-pH-induced unfolding of CcdB …”

Section: Evidence Of Dmg-like Intermediatesmentioning

confidence: 93%

“…Another fluorescence-based method, red-edge excitation shift (REES), has been used to assess the solvent environment surrounding the fluorophore in the protein core. − REES is a special phenomenon where a red shift in the wavelength of the maximum fluorescence emission is observed when excited at the red edge of the excitation spectrum of the fluorophore. In some cases, REES has been observed for DMG-like states. ,− However, unsurprisingly due to the nature of WMGs in which the protein core is hydrated, it is expected to not see any REES. On the other hand, differentiating DMGs from the native state has remained challenging as most global probes fail to distinguish between the native and DMG state if not compared in totality.…”

Section: Distinguishing the Dmg And The Wmg Intermediate Statementioning

confidence: 99%

“…74 Recently, a DMG-like intermediate was observed experimentally during the low-pH-induced unfolding of CcdB. 75 Early evidence for DMG-like intermediates was limited to small single-domain proteins due to the near-native characteristics of DMGs, 60,62,76 which makes DMGs hard to detect using traditional global structural probes. However, in one of our recent studies on human serum albumin (HSA), we demonstrated the first report for the presence of DMG-like equilibrium intermediates for a multidomain protein during low pH-induced unfolding.…”

Section: ■ Evidence Of Dmg-like Intermediatesmentioning

confidence: 99%

See 1 more Smart Citation

Dry Molten Globule-Like Intermediates in Protein Folding, Function, and Disease

Acharya

Jha

2022

J. Phys. Chem. B

View full text Add to dashboard Cite

The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an important field of research for the scientific community for the past five decades. Two important intermediate states, namely, wet molten globule (WMG) and dry molten globule (DMG), have emerged as critical milestones during protein folding−unfolding reactions. While much has been discussed about WMGs as a common unfolding intermediate, the evidence for DMGs has remained elusive owing to their near-native features, which makes them difficult to probe using global structural probes. This Review puts together the available literature and new evidence on DMGs to give a broader perspective on the universality of DMGs and discuss their significance in protein folding, function, and disease.

show abstract

“…CcdB is a homodimeric bacterial toxin that binds to DNA gyrase and causes bacterial cell death. CcdB is an ideal system for understanding the effects of mutation on stability due to its small size (101 amino acids), facile phenotypic readout, and extensive prior experimental characterization [12][13][14] . We screened a number of saturation suppressor libraries of yeast surface displayed CcdB for binding to the DNA Gyrase fragment, GyrA.…”

Section: Introductionmentioning

confidence: 99%

Improved prediction of stabilizing mutations in proteins by incorporation of mutational effects on ligand binding

Ganesan,

Mittal,

Bhat

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

While many computational methods accurately predict destabilizing mutations, identifying stabilizing mutations has remained a challenge, due to their relative rarity. We tested DeltaDeltaG0 predictions from computational predictors such as Rosetta, ThermoMPNN, RaSP, and DeepDDG, using eighty-two mutants of the bacterial toxin CcdB as a test case. On this dataset, the best computational predictor is ThermoMPNN which identifies stabilizing mutations with a precision of 68%. However, the average increase in Tm for these predicted mutations was only 1 degrees C for CcdB, and predictions were poorer for a more challenging target, influenza neuraminidase. Using data from multiple previously described yeast surface display libraries and in vitro thermal stability measurements, we trained logistic regression models to identify stabilizing mutations with a precision of 90% and an average increase in Tm of 3 degrees C for CcdB. When such libraries contain a population of mutants with significantly enhanced binding relative to the corresponding wild type, there is no benefit in using computational predictors. It is then possible to predict stabilizing mutations without any training, simply by examining the distribution of mutational binding scores. This avoids laborious steps of in vitro expression, purification, and stability characterization. When this is not the case, combining data from computational predictors with high-throughput experimental binding data enhances the prediction of stabilizing mutations. However, this requires training on stability data measured in vitro with known stabilized mutants. It is thus feasible to predict stabilizing mutations rapidly and accurately for any system of interest that can be subjected to a binding selection or screen.

show abstract

CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core

Cited by 8 publications

References 73 publications

A look back at the molten globule state of proteins: thermodynamic aspects

A look back at the molten globule state of proteins: thermodynamic aspects

Dry Molten Globule-Like Intermediates in Protein Folding, Function, and Disease

Improved prediction of stabilizing mutations in proteins by incorporation of mutational effects on ligand binding

Contact Info

Product

Resources

About