01762nas a2200169   4500008004100000022001400041245012500055210006900180260007100249300001200320490000600332520106900338100002101407700001901428700002401447856012101471       2016                        eng d  a1948-718500aEvidence for dry molten globule-like domains in the pH-induced equilibrium folding intermediate of a multidomain protein0 aEvidence for dry molten globulelike domains in the pHinduced equ  a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcJAN  a173-1790 v73 a<p>The role of van der Waals (vdW) packing interactions compared to the hydrophobic effect in stabilizing the functional structure of proteins is poorly understood. Here we show, using fluorescence resonance energy transfer, dynamic fluorescence quenching, red-edge excitation shift, and near- and far-UV circular dichroism, that the pH-induced structural perturbation of a multidomain protein leads to the formation of a state in which two out of the three domains have characteristics of dry molten globules, that is, the domains are expanded compared to the native protein with disrupted packing interactions but have dry cores. We quantitatively estimate the energetic contribution of vdW interactions and show that they play an important role in the stability of the native state and cooperativity of its structural transition, in addition to the hydrophobic effect. Our results also indicate that during the pH-induced unfolding, side-chain unlocking and hydrophobic solvation occur in two distinct steps and not in concerted manner, as commonly believed</p>1 aAcharya, Nirbhik1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/evidence-dry-molten-globule-domains-ph-induced-equilibrium-folding-intermediate-001691nas a2200145   4500008004100000245011600041210006900157260000800226300001400234490000800248520112000256100001901376700002401395856012601419       2017                        eng d00aAlternatively packed dry molten globule-like intermediate in the native state ensemble of a multidomain protein0 aAlternatively packed dry molten globulelike intermediate in the   cOCT  a9336-93470 v1213 aIt has been difficult to quantify the degree of side-chain conformational heterogeneity in the native (N) state ensemble of proteins and the relative energetic contributions of the side-chain packing and the hydrophobic effect in protein stability. Here, we show using multiple site specific spectroscopic probes and tools of thermodynamics that the N state ensemble of a multidomain protein contains an equilibrium intermediate (I) whose interdomain region resembles a dry molten globule. In the I state, a tryptophan residue in the interdomain region is alternatively packed, but its secondary structure and intradomain packing are N-like. The I state also has a larger interdomain distance, but the domain-domain interface is dry and molten. Our results indicate that hydrophobic desolvation and side-chain packing are decoupled during protein folding and that interdomain packing interactions have an important energetic contribution in protein stability. Dynamic interconversion between alternatively packed N-like states could be important for multiple allosteric and ligand binding functions of this protein.1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/alternatively-packed-dry-molten-globule-intermediate-native-state-ensemble-multidomain01700nas a2200157   4500008004100000245010000041210006900141260000800210300001600218490000700234520111600241100002101357700001901378700002401397856012101421       2017                        eng d00aDry molten globule-like intermediate during the base-induced unfolding of a multidomain protein0 aDry molten globulelike intermediate during the baseinduced unfol  cNOV  a30207-302160 v193 aThe nature of the initial structural events during the base-induced unfolding of the native (N) state of proteins is poorly understood. Combining site-specific fluorescence resonance energy transfer, size exclusion chromatography, dynamic fluorescence quenching, red-edge excitation shift and circular dichroism spectroscopy, we show here that an early intermediate during the base-induced unfolding of a multidomain protein, i.e., the B form, has features of a dry molten globule. We show that the N (sic) B transition involves protein expansion and loosening of packing of inter-domain helices near domains I and II without the disruption of intra-domain packing or any change in hydration of the inter-domain region which resembles a molten hydrocarbon. Surprisingly, the disruption of inter-domain packing accounts for 40-45% of the total change in free energy of complete unfolding. Our results show that the disruption of van der Waals packing can be decoupled in different regions of a protein and could occur prior to hydrophobic solvation during base-induced unfolding, challenging the existing notion.1 aAcharya, Nirbhik1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/dry-molten-globule-intermediate-during-base-induced-unfolding-multidomain-protein00607nas a2200145   4500008004100000245012500041210006900166260000800235300001600243490000700259100002100266700001900287700002400306856013100330       2019                        eng d00aDry molten globule-like intermediate during the base-induced unfolding of a multidomain protein (vol 19, pg 20307, 2017)0 aDry molten globulelike intermediate during the baseinduced unfol  cSEP  a20499-204990 v211 aAcharya, Nirbhik1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/dry-molten-globule-intermediate-during-base-induced-unfolding-multidomain-protein-vol-19-pg01524nas a2200157   4500008004100000022001400041245009700055210006900152260000800221300001400229490000800243520094500251100001901196700002401215856012701239       2019                        eng d  a1520-610600aSlow motion protein dance visualized using red-edge excitation shift of a buried fluorophore0 aSlow motion protein dance visualized using rededge excitation sh  cFEB  a1256-12640 v1233 a<p>It has been extremely challenging to detect protein structures with a dynamic core, such as dry molten globules, that remain in equilibrium with the tightly packed native (N) state and that are important for a myriad of entropy-driven protein functions. Here, we detect the higher entropy conformations of a human serum protein, using red-edge excitation shift experiments. We covalently introduced a fluorophore inside the protein core and observed that in a subset of native population, the side chains of the polar and buried residues have different spatial arrangements than the mean population and that they solvate the fluorophore on a timescale much slower than the nanosecond timescale of fluorescence. Our results provide direct evidence for the dense fluidity of protein core and show that alternate side-chain packing arrangements exist in the core that might be important for multiple binding functions of this protein.</p>
1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/slow-motion-protein-dance-visualized-using-red-edge-excitation-shift-buried-fluorophore02266nas a2200217   4500008004100000022001400041245010100055210006900156260000800225300001100233490000800244520150100252653003101753653003001784653002801814653002201842100001901864700001701883700002401900856012401924       2021                        eng d  a0301-462200apH-dependent protein stability switch coupled to the perturbed pKa of a single ionizable residue0 apHdependent protein stability switch coupled to the perturbed pK  cJUL  a1065910 v2743 a<p>The contribution of electrostatic interactions in protein stability has not been fully understood. Burial of an ionizable amino acid inside the hydrophobic protein core can affect its ionization equilibrium and shift its pKa differentially in the native (N) and unfolded (U) states of a protein and this coupling between the folding/ unfolding cycle and the ionization equilibria of the ionizable residue can substantially influence the protein stability. Here, we studied the coupling of the folding/unfolding cycle with the ionization of a buried ionizable residue in a multi-domain protein, Human Serum Albumin (HSA) using fluorescence spectroscopy. A pHdependent change in the stability of HSA was observed in the near native pH range (pH 6.0-9.0). The protonation-deprotonation equilibrium of a single thiol residue that is buried in the protein structure was identified to give rise to the pH-dependent protein stability. We quantified the pKa of the thiol residue in the N and the U states. The mean pKa of the thiol in the N state was upshifted by 0.5 units to 8.7 due to the burial of the thiol in the protein structure. Surprisingly, the mean pKa of the thiol in the U state was observed to be downshifted by 1.3 units to 6.9. These results indicate that some charged residues are spatially proximal to the thiol group in the U state. Our results suggest that, in addition to the N state, electrostatic interactions in the U state are important determinants of protein stability.</p>10aelectrostatic interactions10aFluorescence spectroscopy10aThermodynamic stability10aUnfolded proteins1 aMishra, Prajna1 aPatni, Divya1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/ph-dependent-protein-stability-switch-coupled-perturbed-pka-single-ionizable-residue01837nas a2200217   4500008004100000022001400041245008900055210006900144260000800213300001100221490000800232520109800240653003301338653002401371653002701395653001701422653002301439100001901462700002401481856011401505       2022                        eng d  a0301-462200aNative state conformational heterogeneity in the energy landscape of protein folding0 aNative state conformational heterogeneity in the energy landscap  cAPR  a1067610 v2833 aThe native structure of proteins is central to various functions performed by cells. A vital part of the structure-function paradigm of proteins is their inherent flexibility and dynamics. The dynamic interconversion between the conformational substates in the heterogeneous native state basin of the energy landscape enables a single protein molecule to perform multiple functions. The dynamics among the substates are assisted by the motion of different structural elements of a protein out of which side-chains of amino acids hold a significant position due to their involvement in various functions such as molecular recognition and dynamic allostery. This review briefly describes the origin of conformational heterogeneity in the native state ensemble and the motions of different structural modules that assist the equilibrium dynamics of the conformational substates. The review then centers the discussion on conformational heterogeneity due to side-chain movements in proteins, the experimental methods to detect and characterize them and their role in performing multiple functions.10aConformational heterogeneity10aDry molten globules10aEnergy landscape model10aNative state10aSide-chain packing1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/native-state-conformational-heterogeneity-energy-landscape-protein-folding