Protein Charge Transfer Absorption Spectra: An Intrinsic Probe to Monitor Structural and Oligomeric Transitions in Proteins

Ansari, Mohd. Ziauddin; Kumar, Amrendra; Ahari, Dileep; Priyadarshi, Anurag; Lolla, Padmavathi; Bhandari, Rashna; Swaminathan, Rajaram

doi:10.1016/j.bpj.2017.11.3208

Cited by 5 publications

(13 citation statements)

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“…Further PEST fragment being rich in charged amino acids displays protein charge transfer spectra (ProCharTS). Molar extinction coefficient of ProCharTS absorption (250‐800 nm) has been shown to increase when oligomer formation occurs . Our previous experiment with 150 μM PEST sample at pH 3 did not reveal any notable increase in molar extinction coefficient in the ProCharTS region compared to value calculated from slope of Absorbance/Concentration plot suggesting presence of monomeric PEST fragment at all pH tested.…”

Section: Discussionmentioning

confidence: 71%

“…The UV‐Visible absorption spectra of purified PEST Wt and PEST M1 have been described previously . Steady state fluorescence measurements of PEST M1 Trp were carried out at pH 7.4 to explore environment around its indole ring.…”

Section: Resultsmentioning

confidence: 99%

“…The human c‐Myc PEST fragment (PEST Wt) and its Trp mutant (PEST M1) were cloned between Nde 1 and Xho 1 restriction sites in pET21b expression vector, encoding the PEST Wt and PEST M1 residues with a hexahistidine tag fused with C‐terminus. The PEST Wt and M1 were over expressed in Escherichia coli BL21 (DE3) strain and purified using Ni‐NTA agarose beads as described earlier . The purity of PEST fragments were checked by 15% SDS polyacrylamide gel electrophoresis (Figure S2A,C) and mass of purified proteins (Figure S2B,D) was determined using mass spectrometry as reported previously …”

Section: Experimental Methodsmentioning

confidence: 99%

“…All CD spectra were collected from 190 to 260 nm, with bandwidth of 2 nm, scan speed of 100 nm/min and data pitch of 0.1 nm. Calculations of secondary structure content in PEST Wt and M1 under various conditions were done using CDSSTR program as reported previously . For collecting pH dependent CD spectra, 20 μM of PEST Wt, PEST M1 and PEST Wt without His tag were dissolved in different 5 mM buffers (pH 3‐11), containing 1 mM TCEP.…”

Section: Experimental Methodsmentioning

confidence: 99%

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Structure and dynamics at N‐ and C‐terminal regions of intrinsically disordered human c‐Myc PEST degron reveal a pH‐induced transition

Ansari

Swaminathan

2020

Proteins

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We investigated the structure and Brownian rotational motion of the PEST region (201‐268) from human c‐Myc oncoprotein, whose overexpression/dysregulation is associated with various types of cancer. The 77‐residue PEST fragment revealed a large Stokes radius (~3.1 nm) and CD spectrum highlighting abundance of disordered structure. Changes in structure/dynamics at two specific sites in PEST degron were observed using time‐resolved fluorescence spectroscopy by labeling Cys9 near N‐terminal with dansyl probe and inserting a Trp70 near C‐terminal (PEST M1). Trp in PEST M1 at pH 3 was inaccessible to quencher, showed hindered segmental motion and slow global rotation (~30 ns) in contrast to N‐terminal where the dansyl probe was free, exposed with fast global rotation (~5 ns). Remarkably, this large monomeric structure at acidic pH was retained irrespective of ionic strength (0.03‐0.25 M) and partially so in presence of 6 M Gdn.HCl. With gradual increase in pH, a structural transition (~pH 4.8) into a more exposed and freely rotating Trp was noticeable. Interestingly, the induced structure at C‐terminal also influenced the dynamics of dansyl probe near N‐terminal, which otherwise remained unstructured at pH > 5. FRET measurements confirmed a 11 Å decrease in distance between dansyl and indole at pH 4 compared to pH 9, coinciding with enhanced ANS binding and increase in strand/helix population in both PEST fragments. The protonation of glutamate/aspartate residues in C‐terminal region of PEST is implicated in this disorder‐order transition. This may have a bearing on the role of PEST in endocytic trafficking of eukaryotic proteins.

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Section: Discussionmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Experimental Methodsmentioning

confidence: 99%

Section: Experimental Methodsmentioning

confidence: 99%

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Structure and dynamics at N‐ and C‐terminal regions of intrinsically disordered human c‐Myc PEST degron reveal a pH‐induced transition

Ansari

Swaminathan

2020

Proteins

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“…MYC protein is short-lived and has a central PEST domain (enriched in Pro, Glu/Asp, Ser, Thr residues) (19) which contributes to its instability (20). This disordered region (21) has many Ser residues that could be potential targets for pyrophosphorylation by inositol pyrophosphates.…”

Section: Introductionmentioning

confidence: 99%

A pyro-phosphodegron controls MYC polyubiquitination to regulate cell survival

Lolla

Shah

Unnikannan

et al. 2020

Preprint

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The transcription factor MYC regulates cell survival and growth, and its level is tightly controlled in normal cells. Here, we report that serine pyrophosphorylationan enigmatic posttranslational modification triggered by inositol pyrophosphate signaling moleculescontrols MYC levels via regulated protein degradation. We find that endogenous MYC is stabilized and less polyubiquitinated in cells with reduced inositol pyrophosphates. We show that the inositol pyrophosphate 5-IP7 transfers its high-energy beta phosphate moiety to pre-phosphorylated serine residues in the central PEST domain of MYC. Pyrophosphorylation of MYC promotes its interaction with the E3 ubiquitin ligase FBW7, thereby enhancing MYC polyubiquitination and degradation. FBW7 can bind directly to the PEST domain of MYC in a pyrophosphorylationdependent manner. A stabilized, pyrophosphorylation-deficient form of MYC increases cell death during growth stress in untransformed cells, and promotes cell proliferation in response to mitogens. Thus, control of MYC stability through a novel pyro-phosphodegron provides unexpected insight into the regulation of cell survival in response to environmental cues.

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UV–Visible Lysine–Glutamate Dimer Excitations in Protein Charge Transfer Spectra: TDDFT Descriptions Using an Optimally Tuned CAM-B3LYP Functional

2019

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Recent reports of distinctive UV−vis absorption profiles for monomeric proteins rich in charged amino acids that span 250−800 nm have opened up a new label-free optical spectral window for probing biomolecular structure and interactions. Combined experimental-computational studies have revealed that such broad absorption profiles of these proteins arise from photoexcited charge transfer (CT) transitions in spatially proximal charged amino acids such as lysine (Lys) and glutamate (Glu). Here, using time-dependent density functional theory (TDDFT) with an optimally tuned CAM-B3LYP functional, we refine the computed UV−vis spectra for Lys-Glu dimers within protein folds and quantify the percentage CT character of the constituent transitions. The optimally tuned functionals are derived through a careful analysis of the CAM-B3LYP parameter space for Lys-Glu dimers as a function of amino-acid conformation and side chain separation. Our studies reveal that the tuned Lys-Glu dimer spectrum spans 150−650 nm and possesses 5 specific types of CT excitations with diverse and large spatial charge separation length scales of 2− 10 Å. These include inter-/intra-residue peptide backbone to peptide backbone (BB-CT) excitations spanning 160−210 nm, inter-/intra-residue peptide backbone to side chain (BS-CT) excitations spanning 160−260 nm, and side chain to side chain (SS-CT) excitations, which show the broadest absorption range spanning 260−650 nm.

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Protein Charge Transfer Absorption Spectra: An Intrinsic Probe to Monitor Structural and Oligomeric Transitions in Proteins

Cited by 5 publications

References 0 publications

Structure and dynamics at N‐ and C‐terminal regions of intrinsically disordered human c‐Myc PEST degron reveal a pH‐induced transition

Structure and dynamics at N‐ and C‐terminal regions of intrinsically disordered human c‐Myc PEST degron reveal a pH‐induced transition

A pyro-phosphodegron controls MYC polyubiquitination to regulate cell survival

UV–Visible Lysine–Glutamate Dimer Excitations in Protein Charge Transfer Spectra: TDDFT Descriptions Using an Optimally Tuned CAM-B3LYP Functional

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