Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle.

Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular details of substrate binding remain unresolved. Here, we evaluate the role of the solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of asparagine/arginine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are directed through a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.

Screening of candidate substrates and coupling ions of transporters by thermostability shift assays.

Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.

Online analysis and process control in recombinant protein production (review).

Online analysis and control is essential for efficient and reproducible bioprocesses. A key factor in real-time control is the ability to measure critical variables rapidly. Online in situ measurements are the preferred option and minimize the potential loss of sterility. The challenge is to provide sensors with a good lifespan that withstand harsh bioprocess conditions, remain stable for the duration of a process without the need for recalibration, and offer a suitable working range. In recent decades, many new techniques that promise to extend the possibilities of analysis and control, not only by providing new parameters for analysis, but also through the improvement of accepted, well practiced, measurements have arisen.

Online monitoring of biomass accumulation in recombinant yeast cultures.

Methods of biomass monitoring have increasingly been developed towards online, in situ techniques in order to advance process analysis and control. Off-line, ex situ methods, such as dry cell mass determination and direct cell counts, remain the reference for determining cell mass and number, respectively, but this type of analysis is time consuming. Absorbance measurement, which is used extensively as an off-line, ex situ, or online, in situ technique, is fast and straightforward, as the absorbance can be correlated to cell mass and number by a simple calibration. The downside is that absorbance measurements provide no estimation of viability and in situ applications can suffer from interference, such as aeration. Impedance spectroscopy is widely available and provides a quick measure of viable cell concentration, but does not give an estimation of total cell concentration and, hence, potential product. Sensitivity of impedance spectroscopy remains an issue at low cell concentration, and interference during in situ measurements is also a significant factor. In this chapter, a range of protocols is presented for online determination of biomass yields of recombinant yeast cultures.

Monitoring the biomass accumulation of recombinant yeast cultures: offline estimations of dry cell mass and cell counts.

Biomass is one of the most important parameters for process optimization, scale-up and control in recombinant protein production experiments. However, a standard unit of biomass remains elusive. Methods of biomass monitoring have increasingly been developed towards online, in situ techniques in order to advance process analysis and control. Offline, ex situ methods, such as dry cell mass determination and direct cell counts, remain the reference for determining cell mass and number, respectively, but this type of analysis is time consuming. In this chapter, protocols are presented for determining these offline measures of the biomass yield of recombinant yeast cultures.