Computational carbohydrate chemistry: what theoretical methods can tell us

Computational methods have had a long history of application to carbohydrate systems and their development in this regard is discussed. The conformational analysis of carbohydrates differs in several ways from that of other biomolecules. Many glycans appear to exhibit numerous conformations coexisting in solution at room temperature and a conformational analysis of a carbohydrate must address both spatial and temporal properties. When solution nuclear magnetic resonance data are used for comparison, the simulation must give rise to ensemble-averaged properties. In contrast, when comparing to experimental data obtained from crystal structures a simulation of a crystal lattice, rather than of an isolated molecule, is appropriate. Molecular dynamics simulations are well suited for such condensed phase modeling. Interactions between carbohydrates and other biological macromolecules are also amenable to computational approaches. Having obtained a three-dimensional structure of the receptor protein, it is possible to model with accuracy the conformation of the carbohydrate in the complex. An example of the application of free energy perturbation simulations to the prediction of carbohydrate-protein binding energies is presented.     

Oligosaccharide microarrays fabricated on aminooxyacetyl functionalized glass surface for characterization of carbohydrate-protein interaction

Carbohydrate-protein interactions play important biological roles in biological processes. But there is a lack of high-throughput methods to elucidate recognition events between carbohydrates and proteins. This paper reported a convenient and efficient method for preparing oligosaccharide microarrays, wherein the underivatized oligosaccharide probes were efficiently immobilized on aminooxyacetyl functionalized glass surface by formation of oxime bonding with the carbonyl group at the reducing end of the suitable carbohydrates via irreversible condensation. Prototypes of carbohydrate microarrays containing 10 oligosaccharides were fabricated on aminooxyacetyl functionalized glass by robotic arrayer. Utilization of the prepared carbohydrate microarrays for the characterization of carbohydrate-protein interaction reveals that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. The limit of detection (LOD) for lectin ConA on the fabricated carbohydrate microarrays was determined to be approximately 0.008 microg/mL. Inhibition experiment with soluble carbohydrates also demonstrated that the binding affinities of lectins to different carbohydrates could be analyzed quantitatively by determining IC(50) values of the soluble carbohydrates with the carbohydrate microarrays. This work provides a simple procedure to prepare carbohydrate microarray for high-throughput parallel characterization of carbohydrate-protein interaction.     

Applications of ion mobility mass spectrometry for high throughput,high resolution glycan analysis

BackgroundDiverse varieties of often heterogeneous glycans are ubiquitous in nature. They play critical roles in recognition events, act as energy stores and provide structural stability at both molecular and cellular levels. Technologies capable of fully elucidating the structures of glycans are far behind the other ‘-omic’ fields. Liquid chromatography (LC) and mass spectrometry (MS) are currently the most useful techniques for high-throughput analysis of glycans. However, these techniques do not provide full unambiguous structural information and instead the gap in full sequence assignment is frequently filled by a priori knowledge of the biosynthetic pathways and the assumption that these pathways are highly conserved.Scope of the reviewThis comprehensive review details the rise of the emerging analytical technique ion mobility spectrometry (IMS) (coupled to MS) to facilitate the determination of three-dimensional shape: the separation and characterization of isobaric glycans, glyco(peptides/proteins), glycolipids, glycosaminoglycans and other polysaccharides; localization of sites of glycosylation; or interpretation of the conformational change to proteins upon glycan binding.Major conclusionsIMS is a highly promising new analytical route, able to provide rapid isomeric separation (ms timescale) of either precursor or product ions facilitating MS characterization. This additional separation also enables the deconvolution of carbohydrate MS(/MS) information from contaminating ions, improving sensitivity and reducing chemical noise. Derivation of collision cross sections (CCS) from IM-MS(/MS) data and subsequent calculations validate putative structures of carbohydrates from ab initio derived candidates. IM-MS has demonstrated that amounts of specific glycan isomers vary between disease states, which would be challenging to detect using standard analytical approaches.General significanceIM-MS is a promising technique that fills an important gap within the Glycomics toolbox, namely identifying and differentiating the three-dimensional structure of chemically similar carbohydrates and glycoconjugates. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Breaking free from the crystal lattice: Structural biology in solution to study protein degraders

Structural biology offers a versatile arsenal of techniques and methods to investigate the structure and conformational dynamics of proteins and their assemblies. The growing field of targeted protein degradation centres on the premise of developing small molecules, termed degraders, to induce proximity between an E3 ligase and a protein of interest to be signalled for degradation. This new drug modality brings with it new opportunities and challenges to structural biologists. Here we discuss how several structural biology techniques, including nuclear magnetic resonance, cryo-electron microscopy, structural mass spectrometry and small angle scattering, have been explored to complement X-ray crystallography in studying degraders and their ternary complexes. Together the studies covered in this review make a case for the invaluable perspectives that integrative structural biology techniques in solution can bring to understanding ternary complexes and designing degraders.     

Recent developments in structural proteomics for protein structure determination

The major challenges in structural proteomics include identifying all the proteins on the genome-wide scale, determining their structure-function relationships, and outlining the precise three-dimensional structures of the proteins. Protein structures are typically determined by experimental approaches such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. However, the knowledge of three-dimensional space by these techniques is still limited. Thus, computational methods such as comparative and de novo approaches and molecular dynamic simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behavior of proteins. This review summarizes recent developments in structural proteomics for protein structure determination; including instrumental methods such as X-ray crystallography and NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulations.     

Quantitative studies of carbohydrate-protein interaction using functionalized bacterial spores in solution and on chips

Carbohydrate-protein interaction is one of the most important molecular events deemed critical for numerous biological processes. Therefore, understanding this interaction is essential. In this study, we used bacterial spore display techniques to present multiple copies of streptavidin on the surface of spores to explore carbohydrate-protein interaction in solution and on chips. By applying bacterial spores displaying streptavidin, we developed a new method which allows sensitive, versatile, and passive detection of carbohydrate-protein interactions with a 10-fold increase in sensitivity. The linear relationship of interactions between carbohydrates and labeled concanavalin A (con A) in solution and on functionalized bacterial spore chips has also been confirmed. To the best of our knowledge, this is the first example of development and characterization of binding behavior in carbohydrateprotein interactions using bacterial spore-displayed streptavidin. We believe this strategy may enable new high-throughput screening of carbohydrate interactions as well as establish a basis for monitoring inhibitors of carbohydrate-binding proteins when developing new drugs.     

New structural insights into carbohydrate-protein interactions from NMR spectroscopy

Recently developed NMR methods have been applied to discover carbohydrate ligands for proteins and to identify their binding epitopes. The structural details of carbohydrate-protein complexes have also been examined by NMR, providing site-specific information on the architecture, binding selectivity and plasticity of the carbohydrate-binding sites of the proteins. New insights into the conformational behaviour of free and protein-bound glycomimetics pave the way for the design of carbohydrate-based therapeutics. Finally, recent progress towards elucidating the influence of glycosylation on peptide conformation will be of key importance to fully understanding the role of carbohydrates in the function of glycopeptides.     

Integrative Structural Biology in the Era of Accurate Structure Prediction

Characterizing the three-dimensional structure of macromolecules is central to understanding their function. Traditionally, structures of proteins and their complexes have been determined using experimental techniques such as X-ray crystallography, NMR, or cryo-electron microscopy—applied individually or in an integrative manner. Meanwhile, however, computational methods for protein structure prediction have been improving their accuracy, gradually, then suddenly, with the breakthrough advance by AlphaFold2, whose models of monomeric proteins are often as accurate as experimental structures. This breakthrough foreshadows a new era of computational methods that can build accurate models for most monomeric proteins. Here, we envision how such accurate modeling methods can combine with experimental structural biology techniques, enhancing integrative structural biology. We highlight the challenges that arise when considering multiple structural conformations, protein complexes, and polymorphic assemblies. These challenges will motivate further developments, both in modeling programs and in methods to solve experimental structures, towards better and quicker investigation of structure–function relationships.     

Modeling conformational states of proteins with AlphaFold

Many proteins exert their function by switching among different structures. Knowing the conformational ensembles affiliated with these states is critical to elucidate key mechanistic aspects that govern protein function. While experimental determination efforts are still bottlenecked by cost, time, and technical challenges, the machine-learning technology AlphaFold showed near experimental accuracy in predicting the three-dimensional structure of monomeric proteins. However, an AlphaFold ensemble of models usually represents a single conformational state with minimal structural heterogeneity. Consequently, several pipelines have been proposed to either expand the structural breadth of an ensemble or bias the prediction toward a desired conformational state. Here, we analyze how those pipelines work, what they can and cannot predict, and future directions.     

Towards the physical basis of how intrinsic disorder mediates protein function

Intrinsically disordered proteins (IDPs) are an important class of functional proteins that is highly prevalent in biology and has broad association with human diseases. In contrast to structured proteins, free IDPs exist as heterogeneous and dynamical conformational ensembles under physiological conditions. Many concepts have been discussed on how such intrinsic disorder may provide crucial functional advantages, particularly in cellular signaling and regulation. Establishing the physical basis of these proposed phenomena requires not only detailed characterization of the disordered conformational ensembles, but also mechanistic understanding of the roles of various ensemble properties in IDP interaction and regulation. Here, we review the experimental and computational approaches that may be integrated to address many important challenges of establishing a "structural" basis of IDP function, and discuss some of the key emerging ideas on how the conformational ensembles of IDPs may mediate function, especially in coupled binding and folding interactions.     

MOLS sampling and its applications in structural biophysics

This review describes the MOLS method and its applications. This computational method has been developed in our laboratory primarily to explore the conformational space of small peptides and identify features of interest, particularly the minima, i.e., the low energy conformations. A systematic “brute-force” search through the vast conformational space for such features faces the insurmountable problem of combinatorial explosion, whilst other techniques, e.g., Monte Carlo searches, are somewhat limited in their region of exploration and may be considered inexhaustive. The MOLS method, on the other hand, uses a sampling technique commonly employed in experimental design theory to identify a small sample of the conformational space that nevertheless retains information about the entire space. The information is extracted using a technique that is a variant of the self-consistent mean field technique, which has been used to identify, for example, the optimal set of side-chain conformations in a protein. Applications of the MOLS method to understand peptide structure, predict the structures of loops in proteins, predict three-dimensional structures of small proteins, and arrive at the best conformation, orientation, and positions of a small molecule ligand in a protein receptor site have all yielded satisfactory results.     

A survey of the 2006-2009 quartz crystal microbalance biosensor literature

Since the publication of the original review of piezoelectric acoustic sensors in this series there has been a consistent, gradual expansion in the number of published papers using 'quartz crystal microbalances' (QCM). Between 2001 and 2009, the number of QCM publications per annum has increased from 49 to 273, with a two-fold increase in papers per annum between 2004 and 2008. Within the field, comparing the time covered by the current to the previous review, there are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein-protein interactions (40% increase) and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterization of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This paper highlights theoretical and practical aspects of the principles that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells and membrane interfaces.     

Thinking outside the crystal: complementary approaches for examining transporter conformational change

As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present 1?F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.     

Structure, recognition and discrimination in RNA aptamer complexes with cofactors, amino acids, drugs and aminoglycoside antibiotics

Through the use of in vitro selection techniques, a number of RNA aptamers have been selected for their ability to bind ligands with high affinity and specificity. The three-dimensional solution structures of a number of these complexes have been solved within the last 4 years. This review focuses on the structural characterization of the RNA aptamers bound to the cofactors FMN and AMP, the amino acids arginine and citrulline, the drug theophylline and the aminoglycoside antibiotic tobramycin in solution. Analysis of the structural features of these complexes allows the identification of molecular themes in RNA aptamer structure, recognition and discrimination.     

Molecular modeling of noncompetitive antagonists of the NMDA receptor: proposal of a pharmacophore and a description of the interaction mode

Since the three-dimensional structure of the NMDA receptor has not been determined experimentally, indirect computer-assisted molecular modeling techniques appear to be of great usefulness in the characterization of the common pharmacophore of all NMDA receptor noncompetitive antagonists, despite their structural differences. Indeed, the conformational analysis of three different chemical families (MK801, PCP, dexoxadrol and their analogues), has allowed us to visualize the different conformations and configurations of each molecule. Superimposition with configurations 1 and 2 of the MK801 molecule has allowed us to propose active conformations and thereafter a geometrical characterization of the pharmacophore, especially the determination of the orientation of the nitrogen lone pair (NLP) related to the phenyl. On the other hand, electrostatic studies, combined with geometrical features, have allowed us to schematize the interaction mode of an active conformation to the binding site. Finally, studies of the molecular lipophilic potential (MLP) have provided us information on the position of lipophilic and hydrophilic zones of the pharmacophore.     

Fourier transform infrared spectroscopic analysis of protein secondary structures

Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared （FTIR） spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the second- ary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.