Real-time conformational changes and controlled orientation of native proteins inside a protein nanoreactor

Protein conformations play crucial roles in most, if not all, biological processes. Here we show that the current carried through a nanopore by ions allows monitoring conformational changes of single and native substrate-binding domains (SBD) of an ATP-Binding Cassette importer in real-time. Comparison with single-molecule Förster Resonance Energy Transfer and ensemble measurements revealed that proteins trapped inside the nanopore have bulk-like properties. Two ligand-free and two ligand-bound conformations of SBD proteins were inferred and their kinetic constants were determined. Remarkably, internalized proteins aligned with the applied voltage bias, and their orientation could be controlled by the addition of a single charge to the protein surface. Nanopores can thus be used to immobilize proteins on a surface with a specific orientation, and will be employed as nanoreactors for single-molecule studies of native proteins. Moreover, nanopores with internal protein adaptors might find further practical applications in multianalyte sensing devices

Engineering nanopores for studying protein dynamics and interactions

In recent years, nanopores have emerged as a powerful tool to study single molecules. In nanopore experiments, the signal is given by the flux of ions passing through a pore under an applied voltage. Native molecules can be recognized and studied by monitoring the changes in ionic current upon interaction with the nanopore. While DNA sequencing using nanopores is now reality, the use of nanopores to study single proteins has become an important goal. By studying proteins at the single-molecule level, the observation of rare intermediates that are invisible in bulk experiments, becomes possible. In our research group we introduced the Cytolysin A (ClyA) nanopore from the bacteria Salmonella typhi, which is larger than the commonly employed α-hemolysin (αHL) nanopore. Nanopores with larger dimensions allow trapping of single proteins inside their lumen so that the dynamic properties and interactions of the protein can be studied. This PhD thesis uses biological nanopores for the following main goals: - Study protein:DNA interactions - Study protein conformational changes - Develop a nanopore biosensor to detect small analytes - Engineer new functions into a nanopore In the first part of the thesis, the ClyA nanopore is used to probe protein:DNA interactions using the well-studied complex between human thrombin (HT) and thrombin-binding aptamer (TBA) as a model system. Interestingly, nanopore currents are so sensitive that conformational heterogeneities that can not be observed by ensemble experiments are revealed. The binding constants of the protein:DNA complexes could be easily determined and matched well with values observed in literature, indicating the effect of confinement in the nanopore is small. Finally, we describe the forces acting on the protein:DNA complex and provide crucial insights for subsequent protein studies with the ClyA nanopore. In the next part, substrate-binding domains (SBD) 1 and 2 from the Lactococcus lactis ABC importer GlnPQ that bind amino acids are trapped in ClyA nanopores. We observed that substrate-bound and free conformations are reflected in the ionic current signal and could follow their conformational dynamics in real-time. Therefore, we show that nanopores provide an excellent tool to study the dynamics of native proteins at the single-molecule level. Interestingly, because they bind their target substrate with high selectivity, substrate-binding domains could also be used as a nanopore adaptor to detect small analytes in solution. We show that nanomolar concentrations of asparagine and glutamine can be detected using SBD proteins as an adaptor immobilised in the ClyA pore. Moreover, we describe a strategy to improve the recognition between the free and substrate-bound current levels by introducing large aromatic amino acids in the ClyA lumen. This project revealed that both the size and chemical composition of the nanopore lumen can affect protein dynamics and that specific protein-protein interactions could increase or decrease the affinity of SBD2 for glutamine. In the last part or this dissertation, the αHL nanopore is engineered into an artificial molecular machine to assist protein folding. Because they share the same seven-fold symmetry as αHL, the prototypical Escherichia coli GroEL-GroES system was selected. Natively, GroEL/ES mediates protein folding by transducing the energy of ATP binding and hydrolysis into concerted motions. Interestingly, the interaction between GroEL and GroES is mediated only by seven flexible loops in GroES. By grafting these functional loops on top of the αHL pore, we produced a functional co-chaperonin pore that binds and assists in protein folding as efficiently as GroES. The different dynamic steps in the interaction between GroEL and GroES could be followed in real-time by the current signal and showed a distinct pattern in absence or presence of unfolded protein substrate. This work provides important insights for protein studies with nanopores. We demonstrate the potential of sensitive nanopore measurements that could distinguish between subtle heterogeneities and conformational changes. We developed a strategy that has important applications for the detection of small analytes and showed how the current signal could be improved in order to build a nanopore biosensor. In the future, nanopores could be incorporated into a portable device, allowing the fast detection of several analytes. Finally, we provide crucial insights for the development of molecular machines which is an important step towards building artificial biosystems.Summary . . . iii Samenvatting . . . v Abbreviations . . . xi Contents . . . xiii List of publications . . . xxi I Introduction . . . 1 1 Single-molecule studies with biological nanopores . . . 3 1.1 Single-molecule studies . . . 4 1.2 Basic principle of nanopore sensing . . . 5 1.3 Types of nanopores . . . 5 1.3.1 Biological versus solid-state pores . . . 5 1.3.2 The alpha-hemolysin nanopore . . . 8 1.3.3 The ClyA nanopore . . . 8 1.4 Applications of biological nanopore experiments . . . 10 1.4.1 Detection of small molecules . . . 11 1.4.2 Detection of peptides and proteins . . . 12 1.4.3 Nanopore enzymology . . . 15 2 Single-channel recordings in planar lipid bilayers . . . 21 2.1 The experimental set-up . . . 21 2.2 Bilayer formation and nanopore insertion . . . 23 2.3 Data acquisition, filtering and analysis . . . 23 3 Aims . . . 25 II Results . . . 29 4 Detection of two isomeric binding configurations in a protein:aptamer complex with a biological nanopore . . . 31 4.1 Abstract . . . 32 4.2 Introduction . . . 32 4.3 Results . . . 36 4.3.1 Interaction of HT with ClyA nanopores . . . 36 4.3.2 Measuring the affinity binding constant for the HT:TBA complex . . . 36 4.3.3 The HT:TBA complex produces two current blockades . . . 37 4.3.4 Single base substitutions to the TT loop induced one type of HT:TBA current blockades . . . 37 4.3.5 Determination of the aptamer dissociation rate constants . 39 4.4 Discussion . . . 42 4.4.1 TBA binds to HT with two isomeric configurations . . . 42 4.4.2 Electroosmotic and electrophoretic effects on the complex internalization . . . 43 4.4.3 Effect of nanopore confinement on the HT:TBA binding constants . . . 43 4.5 Conclusion . . . 44 4.6 Materials andmethods . . . 45 4.6.1 Preparation of aptamers and human thrombin . . . 45 4.6.2 Expression and purification of ClyA nanopores . . . 45 4.6.3 Electrical recordings in planar lipid bilayers . . . 46 4.6.4 Determination of the thrombin:aptamer complex blockades . . . 46 4.7 Additional remarks . . . 47 5 Conformational dynamics and controlled orientation of single native proteins inside a nanopore . . . 49 5.1 Abstract . . . 50 5.2 Introduction . . . 50 5.3 Results and discussion . . . 52 5.3.1 Conformational dynamics of SBD1 inside ClyA nanopores . . . 52 5.3.2 Conformational dynamics of SBD2 inside ClyA nanopores . . . 53 5.3.3 SBD2 has a fixed orientation inside ClyA that can be controlled . . . 56 5.3.4 SBD1 and SBD2 might adoptmultiple configurations . . . 57 5.4 Conclusion . . . 60 5.5 Materials andmethods . . . 61 5.5.1 Modelling of SBD2 variants . . . 61 5.5.2 Preparation of SBD2 variants . . . 66 5.5.3 Purification of SBD1, SBD2 and variants . . . 67 5.5.4 Expression and purification of ClyA nanopores . . . 67 5.5.5 Electrical recordings in planar lipid bilayers . . . 68 5.5.6 Determination of the kinetic parameters of SBD1 and SBD2 . . . 68 5.5.7 Autocorrelation analysis of SBD1 binding rates . . . 69 6 Engineering ClyA nanopores for enhanced analyte recognition . . . 71 6.1 Abstract . . . 72 6.2 Introduction . . . 72 6.3 Results . . . 74 6.3.1 Systematic screening of SBD2 blockadeswith ClyA-tryptophan variants . . . 74 6.3.2 Effect of tryptophan ring on SBD2 glutamine binding kinetics . . . 77 6.3.3 Effects of amino acid substitutions at ClyA position 56 on SBD2 recognition . . . 80 6.4 Discussion . . . 84 6.4.1 Introduction of bulky residues improves the recognition . . . 84 6.4.2 Effect of confinement on the SBD2 kinetics . . . 85 6.4.3 Residence site of SBD2 in the ClyA pore . . . 86 6.5 Conclusion . . . 87 6.6 Materials andmethods . . . 88 6.6.1 Purification of SBD2 . . . 88 6.6.2 Preparation of ClyA variants . . . 88 6.6.3 Expression and purification of ClyA nanopores . . . 89 6.6.4 Electrical recordings in planar lipid bilayers . . . 89 6.6.5 Data analysis for SBD2 experiments . . . 90 7 Engineering a nanopore with co-chaperonin function . . . 93 7.1 Abstract . . . 94 7.2 Introduction . . . 94 7.3 Results and Discussion . . . 95 7.3.1 Design of a GroES nanopore . . . 95 7.3.2 Characterization of nanopore-GroES chimera proteins . . . 97 7.3.3 Single-channel recordings with a single-ring GroEL . . . 98 7.3.4 GroEL intermediate states . . . 98 7.3.5 Possible mechanism for GroEL induced current blockades . . . 101 7.3.6 Monitoring the SR1 assisted protein refolding with aHLGroESL nanopores . . . 103 7.3.7 Role of GroEL intermediates during chaperonin-mediated protein folding . . . 104 7.4 Conclusions . . . 104 7.5 Materials andmethods . . . 105 7.5.1 Materials . . . 105 7.5.2 Protein preparation . . . 105 7.5.3 Protein assays . . . 106 7.5.4 Electrical recordings from planar lipid bilayers . . . 106 7.5.5 Data analysis . . . 106 7.5.6 Homologymodel of GroEL-®HL-GroESL . . . 107 7.5.7 Sample preparation and negative-stain EM grid preparation . . . 107 7.5.8 EMand two-dimensional image analysis . . . 107 III Conclusions . . . 109 8 Conclusions and perspectives . . . 111 8.1 General conclusions . . . 112 8.2 Future perspectives . . . 116 Bibliography . . . 117 IV Appendices A Supporting information: Detection of two isomeric binding configurations in a protein-aptamer complex with a biological nanopore . . . A-1 A.1 Additional figures . . . A-2 B Supporting information: Conformational dynamics and controlled orientation of single native proteins inside a nanopore . . . B-1 B.1 Additional discussion . . . B-1 B.1.1 Mechanism for the binding of ligands to SBD1 and SBD2 . . . B-1 B.1.2 Ligand-induced binding of glutamine to SBD2 variants . . . B-4 B.2 Additional figures . . . B-6 B.3 Additional tables . . . B-11 C Supporting information: Engineering ClyA nanopores for enhanced analyte recognition . . . C-1 C.1 Additional figures . . . C-2 C.2 Additional tables . . . C-4 D Supporting information: Engineering a nanopore with co-chaperonin function D-1 D.1 Supplementary discussion . . . D-1 D.1.1 Design of the transmembrane co-chaperonin . . . D-1 D.1.2 Voltage dependence of the GroEL:aHL-GroESL interaction . . . D-2 D.2 Additional experimental procedures . . . D-2 D.2.1 Construction of the aHL-GroESS and aHL-GroESL genes . . . D-2 D.2.2 Construction of the GroEL-LLV gene . . . D-3 D.2.3 Construction of the GroEL-398 gene . . . D-4 D.2.4 Construction of the SR1 . . . D-4 D.2.5 Purification of GroEL, GroEL-LLV, GroEL-398 and SR1 . . . D-4 D.2.6 Purification of aHL, aHL-GroESS and aHL-GroESL . . . D-6 D.2.7 Purification of GroES . . . D-6 D.2.8 Haemolytic assay . . . D-6 D.2.9 ATPase assay . . . D-7 D.2.10 MDH refolding assay . . . D-7 D.2.11 LDH refolding assay . . . D-8 D.2.12 GroEL/GroES and GroEL:aHL-GroESL interaction monitored by proteinase K protection . . . D-8 D.3 Additional tables . . . D-9 D.4 Additional figures . . . D-11nrpages: 202status: publishe

Detection of Two Isomeric Binding Configurations in a Protein-Aptamer Complex with a Biological Nanopore

ProteinDNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we showed that folded proteins can be internalized inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalization of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores can be used to probe conformational heterogeneity in protein:DNA interactions

Single-molecule nanopore enzymology

Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology. This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'

Detection of Two Isomeric Binding Configurations in a Protein–Aptamer Complex with a Biological Nanopore

Protein–DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we showed that folded proteins can be internalized inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalization of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores can be used to probe conformational heterogeneity in protein:DNA interactions

Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.status: publishe

Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination