The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c6A

Cytochrome c 6A is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c 6 from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of + 71 mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c 6A from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c 6 from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c 6A and c 6 fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c 6A acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role. © 2011 Elsevier B.V. All rights reserved

Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: A Dissertation

Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function

Sequence Determinants of the Folding Free-Energy Landscape of beta alpha-Repeat Proteins: A Dissertation

The most common structural platform in biology, the βα-repeat classes of proteins, are represented by the (βα)8TIM barrel topology and the α/β/α sandwich, CheY-like topology. Previous studies on the folding mechanisms of several members of these proteins have suggested that the initial event during refolding involves the formation of a kinetically trapped species that at least partially unfolds before the native conformation can be accessed. The simple topologies of these proteins are thought to permit access to locally folded regions that may coalesce in non-native ways to form stable interactions leading to misfolded intermediates. In a pair of TIM barrel proteins, αTS and sIGPS, it has been shown that the core of the off-pathway folding intermediates is comprised of locally connected clusters of isoleucine, leucine and valine (ILV) residues. These clusters of Branched Aliphatic Side Chains (BASiC) have the unique ability to very effectively prevent the penetration of water to the underlying hydrogen bond networks. This property retards hydrogen exchange with solvent, strengthening main chain hydrogen bonds and linking tertiary and secondary structure in a cooperative network of interactions. This property would also promote the rapid formation of collapsed species during refolding. From this viewpoint, the locally connected topology and the appropriate distribution of ILV residues in the sequence can modulate the energy landscapes of TIM barrel proteins. Another sequence determinant of protein stability that can significantly alter the structure and stability of TIM barrels is the long-range main chain-side chain hydrogen bond. Three of these interactions have been shown to form the molecular underpinnings for the cooperative access to the native state in αTS. Global analysis results presented in Chapter II and Chapter III, suggest that the off-pathway mechanism is common to three proteins of the CheY-like topology, namely CheY, NT-NtrC and Spo0F. These results are corroborated by Gō-simulations that are able to identify the minimal structure of kinetically trapped species during the refolding of CheY and Spo0F. The extent of transient, premature structure appears to correlate with the number of ILV side chains involved in a large sequence-local cluster that is formed between the central β-sheet and helices α2, α3 and α4. The failure of Gō-simulations to detect off-pathway species during the refolding of NT-NtrC may reflect the smaller number of ILV side chains in its corresponding hydrophobic cluster. In Chapter IV, comparison of the location of large ILV clusters with the hydrogen exchange protected regions in 19 proteins, suggest that clusters of BASiC residues are the primarily determinants of the stability cores of globular proteins. Although the location of the ILV clusters is sufficient to determine a majority of the protected amides in a protein structure, the extent of protection is over predicted by the ILV cluster method. The survey of 71 TIM barrel proteins presented in Chapter V, suggests that a specific type of long-range main chain-side chain hydrogen bond, termed “βα hairpin clamp” is a common feature in the βα-repeat proteins. The location and sequence patterns observed demonstrate an evolutionary signature of the βαβ modules that are the building blocks of several βα-repeat protein families. In summary, the work presented in this thesis recognizes the role of sequence in modulating the folding free energy landscapes of proteins. The formation of off-pathway folding intermediates in three CheY-like proteins and the differences in the proposed extent of structure formed in off-pathway intermediates of these three proteins, suggest that both topology and sequence play important and concerted roles in the folding of proteins. Locally connected ILV can clusters lead to off-pathway traps, whereas the formation of the productive folding path requires the development of long-range nativelike topological features to form the native state. The ability of ILV clusters to link secondary and tertiary structure formation enables them to be at the core of this cooperative folding process. Very good correlations between the locations of ILV clusters and both strong protection against exchange and the positions of folding nuclei for a variety of proteins reported in the literature support the generality of the BASiC hypothesis. Finally, the discovery of a novel pattern of H-bond interactions in the TIM barrel architecture, between the amide hydrogen of a core ILV residue with a polar side chain, bracketing βαβ modules, suggests a means for establishing cooperativity between different types of side chain interactions towards formation of the native structure. See Additional Files for copies of the source code for the global analysis program and the cluster analysis program

Exploring the Role of Large Clusters of Branched Aliphatic Residues on the Folding Free Energy Landscape of (βα)8 TIM Barrel Proteins

(βα)8 TIM barrel proteins are one of the most common structural motifs found in biology. They have a complex folding free energy landscape that includes an initial off-pathway intermediate as well as two on-pathway intermediates. The formation of these intermediates is hypothesized to be driven by large clusters of the branched chain amino acids, isoleucine, leucine, and valine (ILV). All-atom MD simulations and circular dichroism experiments on polar mutants of the hydrophobic clusters of α-Trp synthase, a TIM barrel protein, revealed the importance of dehydrating the clusters on intermediate states. Custom, single-piece microfluidic chips were interfaced with small angle x-ray scattering and time resolved FRET experiments to monitor the role of a large ILV cluster on the microsecond timescale in a second TIM barrel protein, sIGPS. Dimensional analysis of the initial misfolded intermediate showed an ILV cluster was responsible for the initiation of structure in the intermediate. Early structure formation in the ILV cluster was confirmed by coarse grained simulations. Native state hydrogen exchange experiments were used to probe the higher energy species that are in equilibrium with the native state. Results from the NMR experiment complement the kinetic studies as the core of stability found by NMR mapped back to the same region of the ILV cluster that was found to initiate folding. When taken together, the results show the importance of hydrophobic clusters on the entire free energy surface of TIM barrel proteins

Universality in protein residue networks

Residue networks representing 595 nonhomologous proteins are studied. These networks exhibit universal topological characteristics as they belong to the topological class of modular networks formed by several highly interconnected clusters separated by topological cavities. There are some networks which tend to deviate from this universality. These networks represent small-size proteins having less than 200 residues. We explain such differences in terms of the domain structure of these proteins. On the other hand, we find that the topological cavities characterizing proteins residue networks match very well with protein binding sites. We then investigate the effect of the cutoff value used in building the residue network. For small cutoff values, less than 5Å, the cavities found are very large corresponding almost to the whole protein surface. On the contrary, for large cutoff value, more than 10.0 Å, only very large cavities are detected and the networks look very homogeneous. These findings are useful for practical purposes as well as for identifying "protein-like" complex networks. Finally, we show that the main topological class of residue networks is not reproduced by random networks growing according to Erdös-Rényi model or the preferential attachment method of Barabási-Albert. However, the Watts-Strogatz (WS) model reproduces very well the topological class as well as other topological properties of residue network. We propose here a more biologically appealing modification of the WS model to describe residue networks

Frustration in Biomolecules

Biomolecules are the prime information processing elements of living matter. Most of these inanimate systems are polymers that compute their structures and dynamics using as input seemingly random character strings of their sequence, following which they coalesce and perform integrated cellular functions. In large computational systems with a finite interaction-codes, the appearance of conflicting goals is inevitable. Simple conflicting forces can lead to quite complex structures and behaviors, leading to the concept of "frustration" in condensed matter. We present here some basic ideas about frustration in biomolecules and how the frustration concept leads to a better appreciation of many aspects of the architecture of biomolecules, and how structure connects to function. These ideas are simultaneously both seductively simple and perilously subtle to grasp completely. The energy landscape theory of protein folding provides a framework for quantifying frustration in large systems and has been implemented at many levels of description. We first review the notion of frustration from the areas of abstract logic and its uses in simple condensed matter systems. We discuss then how the frustration concept applies specifically to heteropolymers, testing folding landscape theory in computer simulations of protein models and in experimentally accessible systems. Studying the aspects of frustration averaged over many proteins provides ways to infer energy functions useful for reliable structure prediction. We discuss how frustration affects folding, how a large part of the biological functions of proteins are related to subtle local frustration effects and how frustration influences the appearance of metastable states, the nature of binding processes, catalysis and allosteric transitions. We hope to illustrate how Frustration is a fundamental concept in relating function to structural biology.Comment: 97 pages, 30 figure

Similarities between protein folding and granular jamming.

Grains and glasses, widely different materials, arrest their motions upon decreasing temperature and external load, respectively, in common ways, leading to a universal jamming phase diagram conjecture. However, unified theories are lacking, mainly because of the disparate nature of the particle interactions. Here we demonstrate that folded proteins exhibit signatures common to both glassiness and jamming by using temperature- and force-unfolding molecular dynamics simulations. Upon folding, proteins develop a peak in the interatomic force distributions that falls on a universal curve with experimentally measured forces on jammed grains and droplets. Dynamical signatures are found as a dramatic slowdown of stress relaxation upon folding. Together with granular similarities, folding is tied not just to the jamming transition, but a more nuanced picture of anisotropy, preparation protocol and internal interactions emerges. Results have implications for designing stable polymers and can open avenues to link protein folding to jamming theory

Comparative analysis of rigidity across protein families

We present a comparative study in which 'pebble game' rigidity analysis is applied to multiple protein crystal structures, for each of six different protein families. We find that the main-chain rigidity of a protein structure at a given hydrogen bond energy cutoff is quite sensitive to small structural variations, and conclude that the hydrogen bond constraints in rigidity analysis should be chosen so as to form and test specific hypotheses about the rigidity of a particular protein. Our comparative approach highlights two different characteristic patterns ('sudden' or 'gradual') for protein rigidity loss as constraints are removed, in line with recent results on the rigidity transitions of glassy networks

Biochemical analysis of the W28F mutant of human class Pi glutathione S-transferase

A dissertation submitted in fulfilment of the requirements for the degree of Master of Science at the University of the Witwatersrand. Johannesburg, October 1996.Glutathione S-transferase (GST) class Pi has two tryptophan residues which are conserved within domain one. Trp38 plays a functional role in sequestering glutathione at the active site, whereas Trp28 plays a structural role. The effects of the sterically-conservative substitution of Trp28 to Phe were investigated. When the W28F mutant was compared with the wild-type enzyme, mutation of Ttp28 to Phe was not well tolerated and resulted in a dimeric protein with impaired catalytic function and conformational stability. [Abbreviated Abstract. Open document to view full version]AC201

Elastic properties of proteins: insight on the folding process and evolutionary selection of native structures

We carry out a theoretical study of the vibrational and relaxation properties of naturally-occurring proteins with the purpose of characterizing both the folding and equilibrium thermodynamics. By means of a suitable model we provide a full characterization of the spectrum and eigenmodes of vibration at various temperatures by merely exploiting the knowledge of the protein native structure. It is shown that the rate at which perturbations decay at the folding transition correlates well with experimental folding rates. This validation is carried out on a list of about 30 two-state folders. Furthermore, the qualitative analysis of residues mean square displacements (shown to accurately reproduce crystallographic data) provides a reliable and statistically accurate method to identify crucial folding sites/contacts. This novel strategy is validated against clinical data for HIV-1 Protease. Finally, we compare the spectra and eigenmodes of vibration of natural proteins against randomly-generated compact structures and regular random graphs. The comparison reveals a distinctive enhanced flexibility of natural structures accompanied by slow relaxation times at the folding temperature. The fact that these properties are intimately connected to the presence and assembly of secondary motifs hints at the special criteria adopted by evolution in the selection of viable folds.Comment: Revtex 17 pages, 13 eps figure