Overcoming barriers to membrane protein structure determination

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Novel strategies to improve recombinant membrane protein production in yeast

Approximately 60% of pharmaceuticals target membrane proteins; 30% of the human genome codes for membrane proteins yet they represent less than 1% of known unique crystal structures deposited in the Protein Data Bank (PDB), with 50% of structures derived from recombinant membrane proteins having been synthesized in yeasts. G protein-coupled receptors (GPCRs) are an important class of membrane proteins that are not naturally abundant in their native membranes. Unfortunately their recombinant synthesis often suffers from low yields; moreover, function may be lost during extraction and purification from cell membranes, impeding research aimed at structural and functional determination. We therefore devised two novel strategies to improve functional yields of recombinant membrane proteins in the yeast Saccharomyces cerevisiae. We used human adenosine A2A receptor (hA2AR) as a model GPRC since it is functionally and structurally well characterised.In the first strategy, we investigated whether it is possible to provide yeast cells with a selective advantage (SA) in producing the fusion protein hA2AR-Ura3p when grown in medium lacking uracil; Ura3p is a decarboxylase that catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, generating uridine monophosphate. The first transformant (H1) selected using the SA strategy gave high total yields of hA2AR-Ura3p, but low functional yields as determined by radio-ligand binding, leading to the discovery that the majority of the hA2AR-Ura3p had been internalized to the vacuole. The yeast deletion strain spt3Δ is thought to have slower translation rates and improved folding capabilities compared to wild-type cells and was therefore utilised for the SA strategy to generate a second transformant, SU1, which gave higher functional yields than H1. Subsequently hA2AR-Ura3p from H1 was solubilised with n-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, which yielded functional hA2AR-Ura3p at the highest yield of all approaches used. The second strategy involved using knowledge of translational processes to improve recombinant protein synthesis to increase functional yield. Modification of existing expression vectors with an internal ribosome entry site (IRES) inserted into the 5ˊ untranslated region (UTR) of the gene encoding hA2AR was employed to circumvent regulatory controls on recombinant synthesis in the yeast host cell. The mechanisms involved were investigated through the use of yeast deletion strains and drugs that cause translation inhibition, which is known to improve protein folding and yield. The data highlight the potential to use deletion strains to increase IRES-mediated expression of recombinant hA2AR. Overall, the data presented in this thesis provide mechanistic insights into two novel strategies that can increase functional membrane protein yields in the eukaryotic microbe, S. cerevisiae.

PPD v1.0 - an integrated, web-accessible database of experimentally determined protein pKa values

The Protein pKa Database (PPD) v1.0 provides a compendium of protein residue-specific ionization equilibria (pKa values), as collated from the primary literature, in the form of a web-accessible postgreSQL relational database. Ionizable residues play key roles in the molecular mechanisms that underlie many biological phenomena, including protein folding and enzyme catalysis. The PPD serves as a general protein pKa archive and as a source of data that allows for the development and improvement of pKa prediction systems. The database is accessed through an HTML interface, which offers two fast, efficient search methods: an amino acid-based query and a Basic Local Alignment Search Tool search. Entries also give details of experimental techniques and links to other key databases, such as National Center for Biotechnology Information and the Protein Data Bank, providing the user with considerable background information.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

The synthesis of recombinant membrane proteins in yeast for structural studies

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

Overcoming bottlenecks in the membrane protein structural biology pipeline

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.

Negative data in DEA:a directional distance approach applied to bank branches

This paper is drawn from the use of data envelopment analysis (DEA) in helping a Portuguese bank to manage the performance of its branches. The bank wanted to set targets for the branches on such variables as growth in number of clients, growth in funds deposited and so on. Such variables can take positive and negative values but apart from some exceptions, traditional DEA models have hitherto been restricted to non-negative data. We report on the development of a model to handle unrestricted data in a DEA framework and illustrate the use of this model on data from the bank concerned.

Malmquist-type indices in the presence of negative data:an application to bank branches

In this paper we develop an index and an indicator of productivity change that can be used with negative data. For that purpose the range directional model (RDM), a particular case of the directional distance function, is used for computing efficiency in the presence of negative data. We use RDM efficiency measures to arrive at a Malmquist-type index, which can reflect productivity change, and we use RDM inefficiency measures to arrive at a Luenberger productivity indicator, and relate the two. The productivity index and indicator are developed relative to a fixed meta-technology and so they are referred to as a meta-Malmquist index and meta-Luenberger indicator. We also address the fact that VRS technologies are used for computing the productivity index and indicator (a requirement under negative data), which raises issues relating to the interpretability of the index. We illustrate how the meta-Malmquist index can be used, not only for comparing the performance of a unit in two time periods, but also for comparing the performance of two different units at the same or different time periods. The proposed approach is then applied to a sample of bank branches where negative data were involved. The paper shows how the approach yields information from a variety of perspectives on performance which management can use.

Comparative efficiency analysis of Portuguese bank branches

The advent of Internet banking and phone banking is changing the role of bank branches from a predominantly transaction-based one to a sales-oriented role. This paper reports on an assessment of the branches of a Portuguese bank in terms of their performance in their new roles in three different areas: Their efficiency in fostering the use of new transaction channels, their efficiency in increasing sales and their customer base, and their efficiency in generating profits. Service quality is also a major issue in service organisations like bank branches, and therefore we analyse the way this dimension of performance has been accounted for in the literature and take it into account in our empirical application. We have used data envelopment analysis (DEA) for the different performance assessments, but we depart from traditional DEA models in some cases. Performance comparisons on each dimension allowed us to identify benchmark bank branches and also problematic bank branches. In addition, we found positive links between operational and profit efficiency and also between transactional and operational efficiency. Service quality is positively related with operational and profit efficiency. © 2006 Elsevier B.V. All rights reserved.

A semi-oriented radial measure for measuring the efficiency of decision making units with negative data, using DEA

Data Envelopment Analysis (DEA) is a nonparametric method for measuring the efficiency of a set of decision making units such as firms or public sector agencies, first introduced into the operational research and management science literature by Charnes, Cooper, and Rhodes (CCR) [Charnes, A., Cooper, W.W., Rhodes, E., 1978. Measuring the efficiency of decision making units. European Journal of Operational Research 2, 429–444]. The original DEA models were applicable only to technologies characterized by positive inputs/outputs. In subsequent literature there have been various approaches to enable DEA to deal with negative data. In this paper, we propose a semi-oriented radial measure, which permits the presence of variables which can take both negative and positive values. The model is applied to data on a notional effluent processing system to compare the results with those yielded by two alternative methods for dealing with negative data in DEA: The modified slacks-based model suggested by Sharp et al. [Sharp, J.A., Liu, W.B., Meng, W., 2006. A modified slacks-based measure model for data envelopment analysis with ‘natural’ negative outputs and inputs. Journal of Operational Research Society 57 (11) 1–6] and the range directional model developed by Portela et al. [Portela, M.C.A.S., Thanassoulis, E., Simpson, G., 2004. A directional distance approach to deal with negative data in DEA: An application to bank branches. Journal of Operational Research Society 55 (10) 1111–1121]. A further example explores the advantages of using the new model.

New insights on measuring bank branches efficiency through DEA:transactional, operational, and profit assessments

This thesis presents a number of methodological developments that were raised by a real life application to measuring the efficiency of bank branches. The advent of internet banking and phone banking is changing the role of bank branches from a predominantly transaction-based one to a sales-oriented role. This fact requires the development of new forms of assessing and comparing branches of a bank. In addition, performance assessment models must also take into account the fact that bank branches are service and for-profit organisations to which providing adequate service quality as well as being profitable are crucial objectives. This study analyses bank branches performance in their new roles in three different areas: their effectiveness in fostering the use of new transaction channels such as the internet and the telephone (transactional efficiency); their effectiveness in increasing sales and their customer base (operational efficiency); and their effectiveness in generating profits without compromising the quality of service (profit efficiency). The chosen methodology for the overall analysis is Data Envelopment Analysis (DEA). The application attempted here required some adaptations to existing DEA models and indeed some new models so that some specialities of our data could be handled. These concern the development of models that can account for negative data, the development of models to measure profit efficiency, and the development of models that yield production units with targets that are nearer to their observed levels than targets yielded by traditional DEA models. The application of the developed models to a sample of Portuguese bank branches allowed their classification according to the three performance dimensions (transactional, operational and profit efficiency). It also provided useful insights to bank managers regarding how bank branches compare between themselves in terms of their performance, and how, in general, the three performance dimensions are connected between themselves.

Implications of bank ownership for the credit channel of monetary policy transmission:evidence from India

Using bank-level data from India, we examine the impact of ownership on the reaction of banks to monetary policy, and also test whether the reaction of different types of banks to monetary policy changes is different in easy and tight policy regimes. Our results suggest that there are considerable differences in the reactions of different types of banks to monetary policy initiatives of the central bank, and that the bank lending channel of monetary policy is likely to be much more effective in a tight money period than in an easy money period. We also find differences in impact of monetary policy changes on less risky short-term and more risky medium-term lending. We discuss the policy implications of the findings.

Does the World Bank have any impact on human development in the poorest countries? Some preliminary evidence from Africa

In an attempt to better understand the impact of the World Bank on human development in poor countries, we use cross-country data on African countries for the 1990–2002 period to examine this relationship. The coefficient estimates of our parsimonious fixed-effects models indicate that while loans and grants of the Bank have had a positive impact on some relatively short-term indicators of health and education in an average African country, there is little evidence to suggest that such loans and grants have helped these countries to consolidate on the short-term gains.

Interval data without sign restrictions in DEA

Conventional DEA models assume deterministic, precise and non-negative data for input and output observations. However, real applications may be characterized by observations that are given in form of intervals and include negative numbers. For instance, the consumption of electricity in decentralized energy resources may be either negative or positive, depending on the heat consumption. Likewise, the heat losses in distribution networks may be within a certain range, depending on e.g. external temperature and real-time outtake. Complementing earlier work separately addressing the two problems; interval data and negative data; we propose a comprehensive evaluation process for measuring the relative efficiencies of a set of DMUs in DEA. In our general formulation, the intervals may contain upper or lower bounds with different signs. The proposed method determines upper and lower bounds for the technical efficiency through the limits of the intervals after decomposition. Based on the interval scores, DMUs are then classified into three classes, namely, the strictly efficient, weakly efficient and inefficient. An intuitive ranking approach is presented for the respective classes. The approach is demonstrated through an application to the evaluation of bank branches. © 2013.

Building bank brands:how leadership behavior influences employee commitment

Front line employees are critical to service brand success, as their performance brings brand promises to life. Banking employees, like others, must remain committed to their employers, to live the brand, particularly during periods of economic uncertainty and customer frustration. Employees' commitment influences their brand adoption and brand-supporting behavior during service encounters. Effective leadership fosters employee commitment and brand supporting behaviors. This study examines the nature of employee commitment in banking, distinguishing between affective, continuance and normative commitment. The study explores bank leaders, examining whether initiating structure leader behavior or considerate leader behavior is most effective in encouraging employee commitment. Data from a sample of 438 employees in a leading Irish bank reveals the optimal leadership style for employee commitment. © 2012 Elsevier Inc.