Loop-closure principles in protein folding

Simple theoretical concepts and models have been helpful to understand the folding rates and routes of single-domain proteins. As reviewed in this article, a physical principle that appears to underly these models is loop closure.     

Early events in protein folding

Recent advances have significantly increased the time and spectroscopic resolution of protein folding experiments. We can now study the timescale and nature of polypeptide collapse, and how this correlates with secondary and tertiary structure formation. Studies on ultrafast folding proteins and peptides provide experimental benchmarks on a timescale that overlaps directly with that of molecular dynamics simulations. This makes possible direct tests of both simulations and current models of protein folding.     

Changes of protein stiffness during folding detect protein folding intermediates

Single-molecule force-quench atomic force microscopy (FQ-AFM) is used to detect folding intermediates of a simple protein by detecting changes of molecular stiffness of the protein during its folding process. Those stiffness changes are obtained from shape and peaks of an autocorrelation of fluctuations in end-to-end length of the folding molecule. The results are supported by predictions of the equipartition theorem and agree with existing Langevin dynamics simulations of a simplified model of a protein folding. In the light of the Langevin simulations the experimental data probe an ensemble of random-coiled collapsed states of the protein, which are present both in the force-quench and thermal-quench folding pathways.     

Methods for exploring early events in protein folding.

Progress in understanding dynamic aspects of protein folding relies on the continuing development of methods for obtaining more detailed structural information on the transient conformational ensembles that often appear within microseconds of initiating refolding. Advances in rapid mixing and other time-resolved spectroscopic methods have made it possible to explore some of the earliest stages of folding, including the initial formation of compact states, which is determined by the presence of a sequence-specific kinetic barrier, as well as the 'downhill' folding kinetics after the rate-limiting barrier has been crossed.     

Co-operative interactions during protein folding.

The theory for measuring co-operativity between interactions in proteins by protein engineering experiments is developed by introducing a procedure for analysing increasing orders of synergy in a protein with increasing numbers of residues. The (pairwise) interaction energy (delta 2Gint) between two side-chains may be measured experimentally by a double-mutant cycle consisting of the wild-type protein, the two single mutants and the double mutant. This procedure may be extended to three residues to give a value for delta 3Gint for a triple-mutant cube, and to higher orders using multi-dimensional mutant space. We now show that delta 3Gint is the excess energy of adding all three chains compared with the sum of all the pairwise values of delta 2Gint for each of the constituent double-mutant cycles and the sum of all the single addition energies. This physical interpretation extends to higher orders of mutation. delta nGint (i.e. the interaction energy for n residues), thus, reveals the layers of synergy in interactions as a protein is built up. This procedure is applied to measuring changes in synergy during the refolding of barnase for the triad of salt-linked residues Asp8, Asp12 and Arg110, which are mutated to alanine residues. The value of delta 3Gint in the folded structure is 0.77(+/- 0.06) kcal mol-1 (i.e. the triad is 0.77 kcal mol-1 more stable than expected from the sum of the individual pairwise interactions and single contributions). The value of delta 3Gint is still significant in the transition state for unfolding (0.60(+/- 0.07) kcal mol-1) and in the folding intermediate (0.60(+/- 0.13 kcal mol-1)). These results show that synergistic interactions exist in barnase, in its transition state for unfolding and in a refolding intermediate. A direct measurement of the change of co-operativity between the folded state and the transition state for unfolding shows a decrease of 0.17(+/- 0.04) kcal mol-1, suggesting that the initial stages of protein unfolding may be accompanied by some loosening of structure in parts that still interact. The similar extent of co-operativity in the transition state for unfolding and the intermediate in refolding suggests that the intermediate is homogeneous, at least in the region of the salt-linked triad, as heterogeneity would lower the co-operativity.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Causal links between protein folding in the ER and events along the secretory pathway

The 70-kDa heat shock protein (Hsp70) family comprises the most abundant and important group of molecular chaperones. Hsp70s cooperate with a number of cofactors, which define their functions. We recently reported that a yeast protein, Rot1, is a putative cofactor of BiP, an endoplasmic reticulum (ER)-localized Hsp70. Rot1 is an essential ER membrane protein and may be involved in protein folding. Mutation of the ROT1 gene caused defects in cell wall synthesis and lysis of autophagic bodies. We suggest that Rot1 is required for folding of proteins engaged in these cellular processes.     

Study of early events in the protein folding of villin headpiece using molecular dynamics simulation

Protein folding is scientifically and computationally challenging problem. The early phases of protein folding are interesting due to various events like nascent secondary structure formation, hydrophobic collapse leading to formation of non-native or meta-stable conformations. These events occur within a very short time span of 100ns as compared to total folding time of few microseconds. It is highly difficult to observe these events experimentally due to very short lifetime. Molecular dynamics simulation technique can efficiently probe the detailed atomic level understanding about these events. In the present paper, all atom molecular dynamics simulation trajectory of nearly 200ns was carried out for fully solvated villin headpiece with PME treatment using AMBER 7 package. Initial hydrophobic collapse along with secondary structure formation resulted into formation of partially stable non-native conformations. The formation of secondary structural elements and hydrophobic collapse takes place simultaneously in the folding process.     

The kinetics of side chain stabilization during protein folding

The rate of stabilization of side chains during protein folding has never been carefully studied. Recent developments in labeling proteins with (19)F-labeled amino acids coupled with real-time NMR measurements have allowed such measurements to be made. This paper describes the application of this method to the study of several proteins using 6-(19)F-tryptophan as the reporting group. It is found that these side chains adopt their final stable state at the last stages of the folding process and that the stabilization of side chains into their final conformation is a highly cooperative process. It is also possible to show the presence of intermediates in which the side chains are not correctly packed. The technique should be applicable to many systems.     

Unfolding and conformational distributions during protein precipitation

The association of misfolded proteins, or aggregation, is a critical problem in a number of human diseases as well during the expression, refolding, formulation, and delivery of therapeutic proteins. In this study, we investigate lysozyme precipitation with hydrogen exhange using nuclear magnetic resonance (NMR) and mass spectrometry (MS). We show that MS can reveal the presence of conformational distributions, albeit without the detailed structural information afforded by NMR. Further, we find that increases in precipitant concentration alter the structure and composition of precipitates. The selective unfolding of one portion of the protein in these precipitates is correlated with hydrogen exchange patterns observed under nonprecipitating conditions and in other studies of lysozyme.     

Ultrafast events in the folding of ferrocytochrome c

Laser flash photolysis and stopped-flow methods have been used to study the dynamic events in the micro- to millisecond time bin in the refolding of horse ferrocytochrome c in the full range of guanidine hydrochloride concentration at pH 12.8 (+/-0.1), 22 degrees C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 micros. The chain then undergoes diffusive dynamics-mediated contraction and expansion, in which intrapolypeptide ligands make transient contacts with the heme iron, giving rise to two distinct kinetic phases of approximately 0.4 and approximately 3 micros. Under moderate to absolute refolding conditions, the rates of these processes show little dependence on the denaturant concentration, indicating the absence of structural element in the incipient or the relaxed state. Chain expansion and contraction events continue until the polypeptide finds a stable and supportive transition state. The crossing of this transition barrier, which rate-limits the folding of alkaline ferrocytochrome c, is characterized by a stopped-flow measured time constant of approximately 3 ms in aqueous solvent. Observed kinetics thus implicate no submillisecond folding structure. The folding kinetics is effectively two state in which the unfolded polypeptide first relaxes to an unstructured chain and then crosses over a late rate-limiting barrier to achieve the native conformation. The experimentally observed rates as a function of guanidine hydrochloride concentration have been simulated by numerically calculated microscopic rates of a simple kinetic model that captures the essential features of folding.     

Membrane proteins shape up: understanding in vitro folding

Progress in structural biology has begun to reveal the precise architecture of integral membrane proteins. However, the manner in which these complex structures are achieved remains unclear. Recent developments are starting to shed light on the unfolding and folding of a small but growing number of membrane proteins. Mechanistic details derived from kinetic and thermodynamic experiments now enable comparison of the folding of different membrane proteins and their water-soluble cousins. This work also has important implications for other structural and functional studies of membrane proteins in vitro.     

Funnel-like organization in sequence space determines the distributions of protein stability and folding rate preferred by evolution

To understand the physical and evolutionary determinants of protein folding, we map out the complete organization of thermodynamic and kinetic properties for protein sequences that share the same fold. The exhaustive nature of our study necessitates using simplified models of protein folding. We obtain a stability map and a folding rate map in sequence space. Comparison of the two maps reveals a common organizational principle: optimality decreases more or less uniformly with distance from the optimal sequence in the sequence space. This gives a funnel-shaped optimality surface. Evolutionary dynamics of a sequence population on these two maps reveal how the simple organization of sequence space affects the distributions of stability and folding rate preferred by evolution.     

Determination of intramolecular distance distribution during protein folding on the millisecond timescale

A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed. The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined. The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument). At that time the width of that distribution was rather large (16+/-2 A). The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10(-7) cm(2)/second. In a second slower phase of the refolding transition, the width was reduced to its native value (6+/-4 A).