Is protein folding rate dependent on number of folding stages? Modeling of protein folding with ferredoxin-like fold

Statistical analysis of protein folding rates has been done for 84 proteins with available experimental data. A surprising result is that the proteins with multi-state kinetics from the size range of 50–100 amino acid residues (a.a.) fold as fast as proteins with two-state kinetics from the same size range. At the same time, the proteins with two-state kinetics from the size range 101–151 a.a. fold faster than those from the size range 50–100 a.a. Moreover, it turns out unexpectedly that usually in the group of structural homologs from the size range 50–100 a.a., proteins with multi-state kinetics fold faster than those with two-state kinetics. The protein folding for six proteins with a ferredoxin-like fold and with a similar size has been modeled using Monte Carlo simulations and dynamic programming. Good correlation between experimental folding rates, some structural parameters, and the number of Monte Carlo steps has been obtained. It is shown that a protein with multi-state kinetics actually folds three times faster than its structural homologs.     

Is the unfolded state the Rosetta Stone of the protein folding problem?

Solving the protein folding problem is one of the most challenging tasks in the post genomic era. Identification of folding-initiation sites is very important in order to understand the protein folding mechanism. Detection of residual structure in unfolded proteins can yield important clues to the initiation sites in protein folding. A substantial number of studied proteins possess residual structure in hydrophobic regions clustered together in the protein core. These stable structures can work as seeds in the folding process. In addition, local preferences for secondary structure in the form of turns for beta-sheet initiation and helical turns for alpha-helix formation can guide the folding reaction. In this respect the unfolded states, studied at increasing structural resolution, can be the Rosetta Stone of the protein folding problem.     

Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c

It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol⁻¹, whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol⁻¹. A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.     

How long does it take to equilibrate the unfolded state of a protein?

How long does it take to equilibrate the unfolded state of a protein? The answer to this question has important implications for our understanding of why many small proteins fold with two state kinetics. When the equilibration within the unfolded state U is much faster than the folding, the folding kinetics will be two state even if there are many folding pathways with different barriers. Yet the mean first passage times (MFPTs) between different regions of the unfolded state can be much longer than the folding time. This seems to imply that the equilibration within U is much slower than the folding. In this communication we resolve this paradox. We present a formula for estimating the time to equilibrate the unfolded state of a protein. We also present a formula for the MFPT to any state within U, which is proportional to the average lifetime of that state divided by the state population. This relation is valid when the equilibration within U is very fast as compared with folding as it often is for small proteins. To illustrate the concepts, we apply the formulas to estimate the time to equilibrate the unfolded state of Trp-cage and MFPTs within the unfolded state based on a Markov State Model using an ultra-long 208 microsecond trajectory of the miniprotein to parameterize the model. The time to equilibrate the unfolded state of Trp-cage is ∼100 ns while the typical MFPTs within U are tens of microseconds or longer.     

Is the molten globule a third thermodynamic state of protein? The example of α-lactalbumin

Thermal and denaturant-induced transitions of the acid molten globule state of bovine α-lactalbumin (acid [A] state) are analyzed by scanning calorimetry, titration calorimetry, viscosimetry, and derivative spectroscopy. A denaturant-induced heat effect of the A state is shown by a calorimetric difference titration of the A-state versus unfolded (reduced) α-lactalbumin. However, changes of viscosity and derivative spectra do not parallel the heat effect. At thermal denaturation monitored by derivative spectroscopy and scanning microcalorimetry the presence of a gradual transition in α-lactalbumin A state is shown. The results are consistent with the existence of tertiary interactions in the A state and the absence of a cooperative unfolding transition of the molten globule. The results do not support the idea that the molten globule is a third thermodynamic state. Proteins 30:43–48, 1998. © 1998 Wiley-Liss, Inc.     

What is the minimum number of letters required to fold a protein?

Experimental studies have shown that the full sequence complexity of naturally occurring proteins is not required to generate rapidly folding and functional proteins, i.e. proteins can be designed with fewer than 20 letters. This raises the question of what is the minimum number of amino acid types required to encode complex protein folds? Here, we investigate this issue from three aspects. First, we study the minimum sequence complexity that can reserve the necessary structural information for detection of distantly related homologues. Second, we compare the ability of designing foldable model sequences over a wide range of reduced amino acid alphabets, which find the minimum number of letters that have the similar design ability as 20. Finally, we survey the lower bound of alphabet size of globular proteins in a non-redundant protein database. These different approaches give a remarkably consistent view, that the minimum number of letters required to fold a protein is around ten.     

Förster resonance energy transfer as a probe of membrane protein folding

The folding reaction of a β-barrel membrane protein, outer membrane protein A (OmpA), is probed with F?rster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.     

When does a functional correctly describe both the structure and the energy of the transition state?

Requiring that several properties are well reproduced is a severe test on density functional approximations. This can be assessed through the estimation of joint and conditional success probabilities. An example is provided for a small set of molecules, for properties characterizing the transition states (geometries and energies).     

Function of the phosphatidylinositol transfer protein gene family: is phosphatidylinositol transfer the mechanism of action?

Phosphatidylinositol transfer proteins (PITPs) bind and facilitate the transport of phosphatidylinositol (PI) and phosphatidylcholine between membrane compartments. They are highly conserved proteins, are found in both unicellular and multicellular organisms, and can be present as a single domain or as part of a larger, multi-domain protein. The hallmark of PITP proteins is their ability to sequester PI in their hydrophobic pocket. Ablation or knockdown of specific isoforms in vivo has wide ranging effects such as defects in signal transduction via phospholipase C and phosphoinositide 3-kinase, membrane trafficking, stem cell viability, Drosophila phototransduction, neurite outgrowth, and cytokinesis. In this review, we identify the common mechanism underlying each of these phenotypes as the cooperation between PITP proteins and lipid kinases through the provision of PI for phosphorylation. We propose that recruitment and concentration of PITP proteins at specific membrane sites are required for PITP proteins to execute their function rather than lipid transfer.     

Conformational transitions regulate the exposure of a DNA-binding domain in the RuvBL1–RuvBL2 complex

RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1–RuvBL2 complexes at ∼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1–RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1–RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.     

Role of conserved Fα-helix residues in the native fold and stability of the kinase-inhibited oxy state of the oxygen-sensing FixL protein from Sinorhizobium meliloti

The oxygen-sensing FixL protein from Sinorhizobium meliloti is part of the heme-PAS family of gas sensors that regulate many important signal transduction pathways in a wide variety of organisms. We examined the role of the conserved F_α-9 arginine 200 and several other conserved residues on the proximal F_α-helix in the heme domain of SmFixL* using site-directed mutagenesis in conjunction with UV-visible, EPR, and resonance Raman spectroscopy. The F_α-helix variants R200A, E, Q, H, Y197A, and D195A were expressed at reasonable levels and purified to homogeneity. The R200I and Y201A variants did not express in observable quantities. Tyrosine 201 is crucial for forming the native protein fold of SmFixL* while Y197 and R200 are important for stabilizing the kinase-inhibited oxy state. Our results show a clear correlation between H-bond donor ability of the F_α-9 side chain and the rate of heme autoxidation. This trend in conjunction with crystal structures of liganded BjFixL heme domains, show that H-bonding between the conserved F_α-9 arginine and the heme-6-propionate group contributes to the kinetic stability of the kinase-inactivated, oxy state of SmFixL*.     

Does the inhibition of microvillus protein phosphorylation by lysine explain the activity of the latter on calcium transfer?

• 1.1. Is the activity of l-lysine on calcium absorption related to the fact that its phosphorylation is competitive with that of the microvilli proteins involved in the mineral transfer?
• 2.2. The microvilli proteins phosphorylation is not cyclic GMP-dependent but is actually inhibited by l-lysine, used in general at a 100 mM concentration.
• 3.3. The electrophoresis is followed by an autoradiograph which reveals the existence of a phosphorylated protein with a molecular weight of 140,000 daltons. Another phosphorylable protein, clearly visible in some preparations but only detectable in others, has a molecular weight close to 70,000 daltons.
• 4.4. The inhibition by lysine of the microvilli proteins phosphorylation is not specific to a given protein, but is also observed for phosphorylable cytosolic proteins.
• 5.5. A scheme for calcium transfer is proposed. It involves a protein whose phosphorylation should reduce the membrane permeability to calcium.
• 6.6. The following three attributes of the phosphorylable membrane protein—its molecular weight; the fact that another protein (probably its monomer) is also phosphorylable; its well known capacity for phosphorylation—suggest that this protein might actually be alkaline phosphatase whose correlations in calcium metabolism are well known.

     

The protein folding transition state: what are Phi-values really telling us?

Protein engineering-based studies of the folding transition state have accelerated significantly in the last decade, and more than a half dozen proteins have been subjected to extensive Phi-value analysis. A general picture is emerging from these studies of a transition state in which the large majority of experimentally characterized side chains participate in relatively homogeneous and energetically weak interactions playing only a relatively small role in defining relative folding rates.