On the mechanism of protein fold‐switching by a molecular sensor

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild‐type fold (N) to a circularly‐permuted fold (N′), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order–disorder transition. The nature of the conformational change is expected to be similar from protein to protein; thus, it may be possible to employ AFF as a general method to create optical biosensors. Toward that goal, we test basic aspects of the AFF mechanism using the AFF variant of calbindin D_9k. A simple three‐state model for fold switching holds that N and N′ interconvert through the unfolded state. This model predicts that the fundamental properties of the switch—calcium binding affinity, signal response (i.e., fluorescence change upon binding), and switching rate—can be controlled by altering the relative stabilities of N and N′. We find that selectively destabilizing N or N′ changes the equilibrium properties of the switch (binding affinity and signal response) in accordance with the model. However, kinetic data indicate that the switching pathway does not require whole‐molecule unfolding. The rate is instead limited by unfolding of a portion of the protein, possibly in concert with folding of a corresponding region. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.     

Pressure as a denaturing agent in studies of single-point mutants of an amyloidogenic protein human cystatin c

Recently, we presented a convenient method combining a deuterium‐hydrogen exchange and electrospray mass spectrometry for studying high‐pressure denaturation of proteins (Stefanowicz et al., Biosci Rep 2009; 30:91–99). Here, we present results of pressure‐induced denaturation studies of an amyloidogenic protein—the wild‐type human cystatin C (hCC) and its single‐point mutants, in which Val57 residue from the hinge region was substituted by Asn, Asp or Pro, respectively. The place of mutation and the substituting residues were chosen mainly on a basis of theoretical calculations. Observation of H/D isotopic exchange proceeding during pressure induced unfolding and subsequent refolding allowed us to detect differences in the proteins stability and folding dynamics. On the basis of the obtained results we can conclude that proline residue at the hinge region makes cystatin C structure more flexible and dynamic, what probably facilitates the dimerization process of this hCC variant. Polar asparagine does not influence stability of hCC conformation significantly, whereas charged aspartic acid in 57 position makes the protein structure slightly more prone to unfolding. Our experiments also point out pressure denaturation as a valuable supplementary method in denaturation studies of mutated proteins. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Relocation or duplication of the helix A sequence of T4 lysozyme causes only modest changes in structure but can increase or decrease the rate of folding

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.     

Use of the novel fluorescent amino acid p-cyanophenylalanine offers a direct probe of hydrophobic core formation during the folding of the N-terminal domain of the ribosomal protein L9 and provides evidence for two-state folding

Fluorescence-detected stopped flow measurements are the method of choice for studies of protein folding kinetics. However, the methodology suffers from the limitation that the protein of interest either must contain an intrinsic fluorophore or can tolerate its introduction by mutagenesis. Recently, the cyano (nitrile) analogue of phenylalanine has been proposed for use as a fluorescence analogue. Here we take advantage of this new methodology to monitor the formation of the hydrophobic core during the folding of the N-terminal domain of L9 (NTL9). Phenylalanine 5, which is completely buried in the folded state of NTL9, was replaced with p-cyanophenylalanine (p-cyano-Phe). This derivative reports on the formation of the hydrophobic core. The variant adopts the same fold as wild-type NTL9 and is slightly more stable. Refolding and unfolding were monitored using both guanidine HCl and urea jump experiments. In both cases, plots of the natural log of the observed relaxation rate versus denaturant concentration, so-called chevron plots, exhibited the characteristic V shape expected for two-state folding, and no hint of deviation from linearity was observed at low denaturant concentrations. The stability calculated from the measured folding and unfolding rates is in very good agreement with the value obtained from equilibrium measurements as is the m value. The relative compactness of the transition state for folding as defined by the Tanford beta parameter is identical to that of the wild type. The results illustrate the applicability of p-cyano-Phe analogues in protein folding studies and provide further evidence of two-state folding of NTL9.     

Analyzing pathogenic mutations of C5 domain from cardiac myosin binding protein C through MD simulations

The folding properties of wild type and mutants of domain C5 from cardiac myosin binding protein C have been investigated via molecular dynamics simulations within the framework of a native-centric and coarse-grained model. The relevance of a mutation has been assessed through the shift in the unfolding temperature, the change in the unfolding rate it determines and Phi-values analysis. In a previous paper (Guardiani et al. Biophys J 94:1403-1411, 2008), we performed Kinetic simulations on native contact formation revealing an entropy-driven folding pathway originating near the FG and DE loops. This folding mechanism allowed also a possible interpretation of the molecular impact of the three mutations, Arg14His, Arg28His and Asn115Lys involved in the Familial Hypertrophic Cardiomyopathy. Here we extend that analysis by enriching the mutant pool and we identify a correlation between unfolding rates and the number of native contacts retained in the transition state.     

Folding of Escherichia coli DsbC: characterization of a monomeric folding intermediate

The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC was thermodynamically reversible. The equilibrium folding profile measured by fluorescence excited at 280 nm exhibited a three-state transition profile with a stable folding intermediate formed at 0-2.0 M GdnHCl followed by a second transition at higher GdnHCl concentrations. Sedimentation velocity data revealed dissociation of the dimer to the monomer over the concentration range of the first transition (0-2.0 M). In contrast, fluorescence emission data for DsbC excited at 295 nm showed a single two-state transition. Fluorescence emission data for the equilibrium unfolding of the monomeric G49R mutant, excited at either 295 or 280 nm, indicated a single two-state transition. Data obtained for the dimeric Y52W mutant indicated a strong protein concentration dependence of the first transition but no dependence of the second transition in equilibrium unfolding. This suggests that the fluorescence of Y52W sensitively reports conformational changes caused by dissociation of the dimer. Thus, the folding of DsbC follows a three-state transition model with a monomeric folding intermediate formed in 0-2.0 M GdnHCl. The folding of DsbC in the presence of DTT indicates an important role for the non-active site disulfide bond in stabilizing the conformation of the molecule. Dimerization ensures the performance of chaperone and isomerase functions of DsbC.     

Coarse-grained strategy for modeling protein stability in concentrated solutions. II: phase behavior

We use highly efficient transition-matrix Monte Carlo simulations to determine equilibrium unfolding curves and fluid phase boundaries for solutions of coarse-grained globular proteins. The model we analyze derives the intrinsic stability of the native state and protein-protein interactions from basic information about protein sequence using heteropolymer collapse theory. It predicts that solutions of low hydrophobicity proteins generally exhibit a single liquid phase near their midpoint temperatures for unfolding, while solutions of proteins with high sequence hydrophobicity display the type of temperature-inverted, liquid-liquid transition associated with aggregation processes of proteins and other amphiphilic molecules. The phase transition occurring in solutions of the most hydrophobic protein we study extends below the unfolding curve, creating an immiscibility gap between a dilute, mostly native phase and a concentrated, mostly denatured phase. The results are qualitatively consistent with the solution behavior of hemoglobin (HbA) and its sickle variant (HbS), and they suggest that a liquid-liquid transition resulting in significant protein denaturation should generally be expected on the phase diagram of high-hydrophobicity protein solutions. The concentration fluctuations associated with this transition could be a driving force for the nonnative aggregation that can occur below the midpoint temperature.     

Residue specific resolution of protein folding dynamics using isotope-edited infrared temperature jump spectroscopy

A major difficulty in experimental studies of protein folding is the lack of nonperturbing, residue specific probes of folding. Here, we demonstrate the ability to resolve protein folding dynamics at the level of a single residue using 13C=18O isotope-edited infrared spectroscopy. A single 13C=18O isotopic label was incorporated into the backbone of the 36 residue, three-helix bundle villin headpiece subdomain (HP36). The label was placed in a solvent protected region of the second alpha-helix of the protein. The 13C=18O isotopic label shifted the carbonyl stretching frequency to 1572.1 cm-1 in the folded state, well removed from the 12C=16O band of the unlabeled protein backbone. The unique IR signature of the 13C=18O label was exploited to probe the equilibrium thermal unfolding transition using temperature-dependent FTIR spectroscopy. The folding/unfolding dynamics were monitored using temperature-jump (T-jump) IR spectroscopy. The equilibrium unfolding studies showed conformational changes suggestive of a loss of helical structure in helix 2 prior to the global unfolding of the protein. T-jump relaxation kinetics probing both the labeled site and the 12C=16O band were found to be biphasic with similar relaxation rates. The slow relaxation phase (approximately 2 x 10(5) s-1) corresponds to the global folding transition. The location of the label, a buried position in helix 2, provides an important probe of the origin of the fast relaxation phase (approximately 10(7) s-1). This phase has significant amplitude for the labeled position even though it is well protected from solvent in the folded structure. The fast phase likely represents a rapid pre-equilibrium that involves solvent penetration around the label and possible partial unfolding of helix 2 prior to the global unfolding transition. This work represents the first experimental study of ultrafast folding dynamics with residue specific resolution.     

Selective stabilization of a partially unfolded protein by a metabolite

When proteins fold in vivo, the intermediates that exist transiently on their folding pathways are exposed to the potential interactions with a plethora of metabolites within the cell. However, these potential interactions are commonly ignored. Here, we report a case in which a ubiquitous metabolite interacts selectively with a nonnative conformation of a protein and facilitates protein folding and unfolding process. From our previous proteomics study, we have discovered that Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is not known to bind ATP under native conditions, is apparently destabilized in the presence of a physiological concentration of ATP. To decipher the origin of this surprising effect, we investigated the thermodynamics and kinetics of folding and unfolding of GAPDH in the presence of ATP. Equilibrium unfolding of the protein in urea showed that a partially unfolded equilibrium intermediate accumulates in the presence of ATP. This intermediate has a quaternary structure distinct from the native protein. Also, ATP significantly accelerates the unfolding of GAPDH by selectively stabilizing a transition state that is distinct from the native state of the protein. Moreover, ATP also significantly accelerates the folding of GAPDH. These results demonstrate that ATP interacts specifically with a partially unfolded form of GAPDH and affects the kinetics of folding and unfolding of this protein. This unusual effect of ATP on the folding of GAPDH implies that endogenous metabolites may facilitate protein folding in vivo by interacting with partially unfolded intermediates.     

Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations

Folding reaction of goat alpha-lactalbumin has been studied by stopped-flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the alpha-domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native-state stability of goat alpha-lactalbumin against the guanidine hydrochloride-induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild-type and mutant goat alpha-lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high-temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter-residue contacts and with the water molecule penetration in the 400-K simulation as well as in four of the six 600-K simulations. Disruption of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report.     

Mitochondrial aminoacyl-tRNA synthetase single-nucleotide polymorphisms that lead to defects in refolding but not aminoacylation

Defects in organellar translation are the underlying cause of a number of mitochondrial diseases, including diabetes, deafness, encephalopathy, and other mitochondrial myopathies. The most common causes of these diseases are mutations in mitochondria-encoded tRNAs. It has recently become apparent that mutations in nuclear-encoded components of the mitochondrial translation machinery, such as aminoacyl-tRNA synthetases (aaRSs), can also lead to disease. In some cases, mutations can be directly linked to losses in enzymatic activity; however, for many, their effect is unknown. To investigate how aaRS mutations impact function without changing enzymatic activity, we chose nonsynonymous single-nucleotide polymorphisms (nsSNPs) that encode residues distal from the active site of human mitochondrial phenylalanyl-tRNA synthetase. The phenylalanyl-tRNA synthetase variants S57C and N280S both displayed wild-type aminoacylation activity and stability with respect to their free energies of unfolding, but were less stable at low pH. Mitochondrial proteins undergo partial unfolding/refolding during import, and both S57C and N280S variants retained less activity than wild type after refolding, consistent with their reduced stability at low pH. To examine possible defects in protein folding in other aaRS nsSNPs, we compared the refolding of the human mitochondrial leucyl-tRNA synthetase variant H324Q to that of wild type. The H324Q variant had normal activity prior to unfolding, but displayed a refolding defect resulting in reduced aminoacylation compared to wild type after renaturation. These data show that nsSNPs can impact mitochondrial translation by changing a biophysical property of a protein (in this case refolding) without affecting the corresponding enzymatic activity.     

Studies of effects of macromolecular crowding and confinement on protein folding and protein stability

In cells, proteins execute specific tasks in crowded environments; these environments influence their stability and dynamics. Similarly, for an enzyme molecule encapsulated in an inorganic cavity as in biosensors or biocatalysts, confinement or excluded volume plays an important role in its stability and dynamics. In this article we present results of our experimental and theoretical investigations of the confinement and macromolecular crowding effects on protein. On the experimental side we study the stability of encapsulated cytochrome c against unfolding induced by the presence of denaturants, such as urea. Results show that, as the pore size in which protein is trapped is reduced, protein shows higher stability against denaturant-induced unfolding. On the theoretical side, after reviewing our previous study of the confinement effects on the equilibrium and dynamic properties of protein using a minimalist (two-dimensional lattice, Monte Carlo, Brownian dynamics) model, we have extended the model so that the effects of macromolecular crowding on such properties can be studied. Our simulations show that both folding and unfolding times increase with the number of crowders in solution, however, the equilibrium constant is affected such that the equilibrium is shifted towards the folded state. Furthermore, our results show that, for a fixed number of crowders as the size of crowder (or excluded volume) increases, the average size of protein at equilibrium decreases.     

Molecular basis of co-operativity in protein folding.

The folding/unfolding transition of proteins is a highly co-operative process characterized by the presence of very few or no thermodynamically stable partially folded intermediate states. The purpose of this paper is to present a thermodynamic formalism aimed at describing quantitatively the co-operative folding behavior of proteins. In order to account for this behavior, a hierarchical algorithm aimed at evaluating the folding/unfolding partition function has been developed. This formalism defines the partition function in terms of multiple levels of interacting co-operative folding units. A co-operative folding unit is defined as a protein structural element that exhibits two-state folding/unfolding behavior. At the most fundamental level are those structural elements that behave co-operatively as a result of purely local interactions. Higher-order co-operative folding units are formed through interactions between different structural elements. The hierarchical formalism utilizes the crystallographic structure of the protein as a template to generate partially folded conformations defined in terms of co-operative folding units. The Gibbs free energy of those states and their corresponding statistical weights are then computed using experimental energetic parameters determined calorimetrically. This formalism has been applied to the case of myoglobin. It is shown that the hierarchical partition function correctly predicts the presence, energetics and co-operativity of the heat and cold denaturation transitions. The major contribution to the co-operative folding behavior arises from the solvent exposure of non-polar residues located in regions complementary to those that have undergone unfolding. This entropically uncompensated and energetically unfavorable solvent exposure characterizes all partially folded states but not the unfolded state, thus minimizing the population of partially folded intermediates throughout the folding/unfolding transition.     

Thermal unfolding simulations of a multimeric protein--transition state and unfolding pathways

The folding of an oligomeric protein poses an extra challenge to the folding problem because the protein not only has to fold correctly; it has to avoid nonproductive aggregation. We have carried out over 100 molecular dynamics simulations using an implicit solvation model at different temperatures to study the unfolding of one of the smallest known tetramers, p53 tetramerization domain (p53tet). We found that unfolding started with disruption of the native tetrameric hydrophobic core. The transition state for the tetramer to dimer transition was characterized as a diverse ensemble of different structures using Phi value analysis in quantitative agreement with experimental data. Despite the diversity, the ensemble was still native-like with common features such as partially exposed tetramer hydrophobic core and shifts in the dimer-dimer arrangements. After passing the transition state, the secondary and tertiary structures continued to unfold until the primary dimers broke free. The free dimer had little secondary structure left and the final free monomers were random-coil like. Both the transition states and the unfolding pathways from these trajectories were very diverse, in agreement with the new view of protein folding. The multiple simulations showed that the folding of p53tet is a mixture of the framework and nucleation-condensation mechanisms and the folding is coupled to the complex formation. We have also calculated the entropy and effective energy for the different states along the unfolding pathway and found that the tetramerization is stabilized by hydrophobic interactions.     

Bacteriorhodopsin folds into the membrane against an external force

Despite their crucial importance for cellular function, little is known about the folding mechanisms of membrane proteins. Recently details of the folding energy landscape were elucidated by atomic force microscope (AFM)-based single molecule force spectroscopy. Upon unfolding and extraction of individual membrane proteins energy barriers in structural elements such as loops and helices were mapped and quantified with the precision of a few amino acids. Here we report on the next logical step: controlled refolding of single proteins into the membrane. First individual bacteriorhodopsin monomers were partially unfolded and extracted from the purple membrane by pulling at the C-terminal end with an AFM tip. Then by gradually lowering the tip, the protein was allowed to refold into the membrane while the folding force was recorded. We discovered that upon refolding certain helices are pulled into the membrane against a sizable external force of several tens of picoNewton. From the mechanical work, which the helix performs on the AFM cantilever, we derive an upper limit for the Gibbs free folding energy. Subsequent unfolding allowed us to analyze the pattern of unfolding barriers and corroborate that the protein had refolded into the native state.     

The long and short flavodoxins: II. The role of the differentiating loop in apoflavodoxin stability and folding mechanism

Flavodoxins are classified in two groups according to the presence or absence of a approximately 20-residue loop of unknown function. In the accompanying paper (36), we have shown that the differentiating loop from the long-chain Anabaena PCC 7119 flavodoxin is a peripheral structural element that can be removed without preventing the proper folding of the apoprotein. Here we investigate the role played by the loop in the stability and folding mechanism of flavodoxin by comparing the equilibrium and kinetic behavior of the full-length protein with that of loop-lacking, shortened variants. We show that, when the loop is removed, the three-state equilibrium thermal unfolding of apoflavodoxin becomes two-state. Thus, the loop is responsible for the complexity shown by long-chain apoflavodoxins toward thermal denaturation. As for the folding reaction, both shortened and wild type apoflavodoxins display three-state behavior but their folding mechanisms clearly differ. Whereas the full-length protein populates an essentially off-pathway transient intermediate, the additional state observed in the folding of the shortened variant analyzed seems to be simply an alternative native conformation. This finding suggests that the long loop may also be responsible for the accumulation of the kinetic intermediate observed in the full-length protein. Most revealing, however, is that the influence of the loop on the overall conformational stability of apoflavodoxin is quite low and the natively folded shortened variant Delta(120-139) is almost as stable as the wild type protein. The fact that the loop, which is not required for a proper folding of the polypeptide, does not even play a significant role in increasing the conformational stability of the protein supports our proposal (36) that the differentiating loop of long-chain flavodoxins may be related to a recognition function, rather than serving a structural purpose.     

Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants.

The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.     

Unfolding a linker between helical repeats

In many multi-repeat proteins, linkers between repeats have little secondary structure and place few constraints on folding or unfolding. However, the large family of spectrin-like proteins, including alpha-actinin, spectrin, and dystrophin, share three-helix bundle, spectrin repeats that appear in crystal structures to be linked by long helices. All of these proteins are regularly subjected to mechanical stress. Recent single molecule atomic force microscopy (AFM) experiments demonstrate not only forced unfolding but also simultaneous unfolding of tandem repeats at finite frequency, which suggests that the contiguous helix between spectrin repeats can propagate a cooperative helix-to-coil transition. Here, we address what happens atomistically to the linker under stress by steered molecular dynamics simulations of tandem spectrin repeats in explicit water. The results for alpha-actinin repeats reveal rate-dependent pathways, with one pathway showing that the linker between repeats unfolds, which may explain the single-repeat unfolding pathway observed in AFM experiments. A second pathway preserves the structural integrity of the linker, which explains the tandem-repeat unfolding event. Unfolding of the linker begins with a splay distortion of proximal loops away from hydrophobic contacts with the linker. This is followed by linker destabilization and unwinding with increased hydration of the backbone. The end result is an unfolded helix that mechanically decouples tandem repeats. Molecularly detailed insights obtained here aid in understanding the mechanical coupling of domain stability in spectrin family proteins.