Secondary structure of globular proteins at the early and the final stages in protein folding

The ellipticities for an early transient intermediate in refolding observed by kinetic circular dichroism measurements at 220–225 nm for 14 different proteins are summarized, and the ellipticity values are compared with those for the final native proteins and also with the ellipticities expected from a physical theory of protein and polypeptide secondary structure. The results show that a substantial part of the protein secondary structure is in general formed in the earliest detectable intermediate in refolding and that the ellipticities in both the native and the intermediate states are consistent with the physical theory of protein secondary structure

An early immunoreactive folding intermediate of the tryptophan synthase β2 subunit is a ‘molten globule’

The refolding kinetics of the tryptophan synthase β2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured β2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded β2 the fluorescence of ANS passes through a maximum after about 1 s and then ‘slowly’ decreases. These results show the accumulation, in the 1–10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the ‘molten globule’ state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early imuno-reactive intermediate recognized by a monoclonal antibody (t 12 s), it is shown that this native-like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains

Proof-of-Principle Experiment for FEL-Based Coherent Electron Cooling,”

Abstract Coherent electron cooling (CEC) has a potential to significantly boost luminosity of high-energy, highintensity hadron-hadron and electron-hadron colliders. In a CEC system, a hadron beam interacts with a cooling electron beam. A perturbation of the electron density caused by ions is amplified and fed back to the ions to reduce the energy spread and the emittance of the ion beam. To demonstrate the feasibility of CEC we propose a proof-of-principle experiment at RHIC using SRF linac. In this paper, we describe the setup for CeC installed into one of RHIC's interaction regions. We present results of analytical estimates and results of initial simulations of cooling a gold-ion beam at 40 GeV/u energy via CeC

All-or-none Solvent-induced Transitions Between Native, Molten Globule and Unfolded States in Globular Proteins

Backgound: It has long been established that temperature-induced melting of small globular proteins is an all-or-none transition. Little was known, however, about the degree of cooperativity of denaturant-induced transitions in proteins, especially in those cases in which the proteins unfold through the molten globule state. Results; We have processed data on the equilibrium urea-induced and guanidinium chloride (GdmCl)-induced unfolding of globular proteins from the native to the unfolded state, from the native to the molten globule state and from the molten globule to the unfolded state. We show that in all these cases, the cooperativity of unfolding increases linearly with the increase of the molecular weight of the protein up to 25–30 kDa. Conclusion; The cooperative unit of the urea-induced and GdmCl-induced equilibrium transitions of small proteins between the native, molten globule and unfolded states includes the protein molecule as a whole. In other words, both native and molten globule proteins are unfolded by strong denaturing solvents according to an all-or-none mechanism

Further Evidence on the Equilibrium “Pre-molten Globule State”: Four-state Guanidinium Chloride-induced Unfolding of Carbonic Anhydrase B at Low Temperature

Equilibrium guanidinium chloride-induced unfolding of bovine carbonic anhydrase NB has been investigated by a combination of optical methods with size-exclusion chromatography. It has been shown that, as in the case of staphylococcal β-lactamase, bovine carbonic anhydrase B unfolds at low temperature through two equilibrium intermediates; the molten globule and the pre-molten globule states. This pre-molten globule state has a hydrodynamic volume no more than twofold larger than that of the native state, i.e. is relatively compact. It has a pronounced far UV CD spectrum, suggesting the presence of a substantial secondary structure. It binds 8-anilinonaphthalene-1-sulphonate (though weaker than the molten globule state), which suggests the formation of solvent-exposed clusters of non-polar groups. Thus, this novel state of protein molecules shares a number of properties with the “burst” kinetic intermediate of protein folding and can be considered as its equilibrium counterpart

Partly Folded State, a New Equilibrium State of Protein Molecules: Four-state Guanidinium Chloride-induced Unfolding of .Beta.-lactamase at Low Temperature

The Guanidinium chloride (GdmCl)-induced unfolding of β-lactamase has been investigated using a combination of size-exclusion chromatography (SEC-FPLC) and traditional optical methods. It has been shown that at low temperatures, this protein unfolds through two equilibrium intermediates. The first of these intermediates is the molten globule state, while the other (which we have called a partly folded state) is less compact than the molten globule but much more compact than the unfolded state. It also preserves a substantial part of the secondary structure of the native or molten globule state

Kinetic and Equilibrium Folding Intermediates

Our recent experiments on the molten globule state and other protein folding intermediates lead to following conclusions: (i) the molten globule is separated by intramolecular first-order phase transitions from the native and unfolded states and therefore is a specific thermodynamic state of protein molecules; (ii) the novel equilibrium folding intermediate (the ‘pre-molten globule’ state) exists which can be similar to the ‘burst’ kinetic intermediate of protein folding; (iii) proteins denature and release their non-polar ligands at moderately low pH and moderately low dielectric constant, i.e. under conditions which may be related to those near membranes

The ‘molten globule’ state is involved in the translocation of proteins across membranes?

AbstractStrong evidence exists that the translocation of proteins across a variety of membranes involves a non-native or denatured conformational states. On the other hand a compact state having secondary but not rigid tertiary structure and called the ‘molten globule’ state has been identified as being stable under mild denaturing conditions. A similar state has been shown to accumulate on the folding pathway of globular proteins. These states are compact though sufficiently expanded to include water, and they are internally mobile. It is proposed that these molten globule states may be suitable candidates for protein translocation across biological membranes