Mapping the distribution of packing topologies within protein interiors shows predominant preference for specific packing motifs

<p>Abstract</p> <p>Background</p> <p>Mapping protein primary sequences to their three dimensional folds referred to as the 'second genetic code' remains an unsolved scientific problem. A crucial part of the problem concerns the geometrical specificity in side chain association leading to densely packed protein cores, a hallmark of correctly folded native structures. Thus, any model of packing within proteins should constitute an indispensable component of protein folding and design.</p> <p>Results</p> <p>In this study an attempt has been made to find, characterize and classify recurring patterns in the packing of side chain atoms within a protein which sustains its native fold. The interaction of side chain atoms within the protein core has been represented as a contact network based on the surface complementarity and overlap between associating side chain surfaces. Some network topologies definitely appear to be preferred and they have been termed 'packing motifs', analogous to super secondary structures in proteins. Study of the distribution of these motifs reveals the ubiquitous presence of typical smaller graphs, which appear to get linked or coalesce to give larger graphs, reminiscent of the nucleation-condensation model in protein folding. One such frequently occurring motif, also envisaged as the unit of clustering, the three residue clique was invariably found in regions of dense packing. Finally, topological measures based on surface contact networks appeared to be effective in discriminating sequences native to a specific fold amongst a set of decoys.</p> <p>Conclusions</p> <p>Out of innumerable topological possibilities, only a finite number of specific packing motifs are actually realized in proteins. This small number of motifs could serve as a basis set in the construction of larger networks. Of these, the triplet clique exhibits distinct preference both in terms of composition and geometry.</p

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

Similaridade, docking e dinâmica molecular : combinação de estratégias na busca de novos inibidores da hAChE

Tese (doutorado)—Universidade de Brasília, Instituto de Física, Programa de Pós-Graduação em Física, 2017.A doença de Alzheimer é a forma mais comum de demência irreversível entre as pessoas idosas. A diminuição dos níveis de acetilcolina no cérebro de pacientes com a doença está relacionada com a fisiopatologia da doença. A hipótese colinérgica é baseada no aumento do nível de acetilcolina pela inibição reversível da enzima acetilcolinesterase. O objetivo principal deste trabalho é abordar o desempenho de métodos de triagem virtuais com base no ligante e na estrutura da proteína afim de encontrar novos candidatos a inibidores da acetilcolinesterase. Além disso, um protocolo foi desenvolvido para identificar e propor novos inibidores promissores da AChE a partir do banco de dados ZINC com 10 milhões de compostos disponíveis comercialmente. Neste sentido, busca por similaridade 3D usando similaridade por forma e eletrostática além do método de docagem para uma série de compostos foram utilizados para recuperar inibidores da AChE a partir de uma coleção de banco de dados. Simulação por dinâmica molecular foi realizada para estudar os melhores compostos docados. Alguns resíduos importantes foram identificados como indispensáveis para formação do duplo modo de interação entre os compostos selecionados e o sítio de interação da enzima. Todos os resultados indicam o relevante uso do método de similaridade eletrostática combinado com estratégias de docagem para identificar novos possíveis inibidores, e sete novas estruturas foram selecionadas como potentes candidatos anticolinesterásicos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Alzheimer's disease is the most common cause of irreversible dementia among the elderly. The decrease of acetylcholine levels in the brain of patients with the disease is related to the pathophysiology of the disease. The cholinergic hypothesis is based on increasing the level of acetylcholine by the reversible inhibition of the enzyme acetylcholinesterase. The main purpose of this study is to address the performance of virtual screening methods based on ligands and the protein structure of acetylcholinesterase in order to retrieve novel hAChE inhibitors. In addition, a protocol was developed to identify novel hit compounds and propose new promising AChE inhibitors from ZINC database with 10 million commercially available compounds. In this sense, 3D similarity searches using Rapid Overlay of Chemical Structures and similarity analysis through comparison of electrostatic overlay of docked hits were used to retrieve AChE inhibitors from collected databases. Molecular dynamics simulation of 100 ns was carried out to study the best docked compounds from similarity search. Some key residues are identified as crucial for the dual binding mode of inhibitor with interaction site. All results indicated the relevant use of EON and docking strategy for identifying novel hit compounds as promising potential anticholinesterase candidates, and seven new structures were selected as potential hAChE inhibitors

The characterisation and prediction of protein-protein interfaces.

Understanding how proteins interact with each other is of fundamental importance and is one of the most important goals of molecular biology. In order to study the characteristics of protein-protein interaction sites datasets of non-homologous protein-complexes have been compiled. These datasets include 142 obligate homocomplexes, 20 obligate hetero-complexes, 20 enzyme-inhibitor complexes, 15 antibody-antigen complexes, and 10 signaling complexes. Overall, the protein-protein interfaces of obligate complexes were found to be closely packed, relatively hydrophobic when compared to the entire protein exterior, planar, and enriched in residues such as tyrosine, phenylalanine, and isoleucine. In comparison to the protein-protein interfaces found within obligate protein-complexes the protein-protein interfaces of non-obligate protein-complexes were found to be on average much smaller in size and contain larger numbers of polar and charged residues. The bulk properties of the obligate and non-obligate protein-complexes are also discussed. A neural network was used together with the Patch Analysis method of Jones and Thornton (1997) to predict the location of the protein-protein interfaces in selected datasets of obligate homo and hetero-complexes. The Patch Analysis method is based upon defining a series of contiguous patches over the surface of a protein. The physical and chemical characteristics of each patch are encoded in the form of six parameters. One of these parameters is hydrophobicity. Another parameter that is used is accessible surface area (ASA). By comparing average values of these six parameters for the residues in a given surface patch with those covering known protein-protein interfaces the likelihood of a patch corresponding to a protein-protein interface can be assessed. Based upon the results for a dataset of 76 homo-dimers the use of a neural network enhances the accuracy of the original Patch Analysis method by some thirteen percent

Single-Molecule Measurements of Complex Molecular Interactions in Membrane Proteins using Atomic Force Microscopy

Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage

Investigation of Heterogeneous Proteins and Protein Complexes with Native Ion Mobility-Mass Spectrometry and Theory

Native ion mobility-mass spectrometry (IM-MS) offers many advantages for the study of biomolecules and their complexes. High mass accuracy and sensitivity enable unambiguous determination of complex stoichiometries with respect to subunit composition as well as bound ligands. Ion mobility spectrometry adds an additional dimension of separation and can provide some structural information. Native IM-MS experiments are also fast with minimal sample requirements. Because of these reasons, native IM-MS has become an important tool in structural biology, able to investigate challenging samples that may not be amenable to study by other techniques. However, there are still some major challenges for using native IM-MS in the study of biomolecules. Heterogeneity—arising from the presence of multiple conformations, subunit compositions, ligands and small molecules, for example—results in complicated native mass spectra that can be difficult or even impossible to deconvolute and interpret. Characterizing the heterogeneity of these samples is desirable, as reports of lipids, small drugs, and metals being important for physiological structure and function continue to accumulate. Additionally, interpretation of structural information from IM data has remained largely qualitative, and more fundamental questions about this technique persist, including detailed understanding of the nature of gas-phase protein structure and behavior and how it might differ from solution-phase. Investigation into this aspect is required to make structural interpretation from native IM-MS data quantitative. In the first half of this dissertation, strategies to overcome the challenges of heterogeneity are explored, and computational methods are developed to solve the quantitation problem. With these methods, key features of gas-phase protein ion compaction are revealed, allowing more informed interpretation of structural details from this technique. The second half of this dissertation illustrates the wealth of information that can be accessed for challenging, heterogeneous biomolecules in native IM-MS experiments upon application of these computational methods. With results from both experiment and computation, oligomeric states of the membrane pore-forming protein toxin Cytolysin A are identified, and the composition and topology of multimeric β-crystallin protein complexes, which are implicated in cataract formation, are characterized. This dissertation includes previously published and unpublished co-authored material

Periplasmic mechano-transduction networks

Unlike the inner-membrane, the outer-membrane of Gram-negative bacteria cannot be energised by a proton gradient and the periplasmic space is absent of ATP. This poses a significant problem for active processes such as transport against concentration gradients. TonB-dependent transporters (TBDT) are a class of outer-membrane proteins responsible for the scavenging and import of scarce metallo-organic complexes from the environment. They are structurally characterized by a 22-stranded β-barrel with the lumen occluded by an N-terminal globular ‘plug’ domain which contains a conserved binding motif known as the Ton box. Upon substrate binding, the Ton box becomes exposed to the periplasmic space where it forms a non-covalent complex with the C-terminus of a cytoplasmic membrane protein, TonB. The transport process requires energy from a proton motive force coupled with an inner membrane complex TonB-ExbB-ExbD. Regardless of a wealth of structural information, current models of the TonB-dependent transport mechanism are speculative. Conversely, despite no current experimental evidence, it is generally accepted that the plug domain must undergo a large conformational change facilitated by mechanical force exerted onto the Ton box tether by TonB. In this thesis, force spectroscopy, protein engineering, molecular dynamics and bacterial growth assays are used to investigate the effects of force on TonB:TBDT complexes from E. coli. These experiments demonstrate that the channel of the vitamin B12 transporter (BtuB), reconstituted into synthetic liposome, can be opened by the application of force onto the plug domain via the non-covalent binding partner, TonB. Using wild-type BtuB and several of its mutants together with a related receptor (FhuA), the extent of plug remodelling is found to be highly controlled and determined by the cargo the receptor has evolved to transport. For both receptors, the plug domain can be regarded as comprising a mechanically weak channel forming sub-domain, and a mechanically strong sub-domain used both for allosteric signalling and to limit the size of the channel to allow passage of molecules no larger than its cargo. Alongside these findings, structural and biophysical analysis of the periplasmic spanning protein TonB reveals conformation within the proline-rich linker domain, which allows speculation of the origin of the pulling force used for plug remodelling

Protein Structure

Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic