| 1. |
- Abramsson, Mia L, et al.
(author)
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Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometry
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Other publication (other academic/artistic)abstract
- The role of ionizable side chains in the electrospray ionization mass spectrometry of intact proteins remains hotly debated but has not been conclusively addressed because multiple chargeable sites are present in virtually all proteins. Using engineered soluble proteins, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, whilst co-existing conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. We conclude that the sum of charges is governed solely by Coulombic terms, while their locations affect the stability of the protein in the gas phase.
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| 2. |
- Abramsson, Mia L., et al.
(author)
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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry
- 2021
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In: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:12, s. 2385-2393
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Journal article (peer-reviewed)abstract
- In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.
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| 3. |
- Gavrilov, Yulian, et al.
(author)
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Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2
- 2023
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In: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 32:4
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Journal article (peer-reviewed)abstract
- Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics, and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1°C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well-ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes.
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| 4. |
- Ghasriani, Houman, et al.
(author)
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Solution structures of human and porcine beta-microseminoprotein
- 2006
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In: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 362:3, s. 502-515
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Journal article (peer-reviewed)abstract
- beta-Microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs. (c) 2006 Elsevier Ltd. All rights reserved.
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| 5. |
- Hervø-Hansen, Stefan, et al.
(author)
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Charge Interactions in a Highly Charge-Depleted Protein
- 2021
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:6, s. 2500-2508
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Journal article (peer-reviewed)abstract
- Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.
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| 6. |
- Iesmantavicius, Vytautas, et al.
(author)
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Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding
- 2014
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In: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 53:6, s. 1548-1551
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Journal article (peer-reviewed)abstract
- Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence-monitored stopped-flow kinetic measurements we show that the secondary structure content in helix1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.
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| 7. |
- Karlsson, Elin, 1992-
(author)
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Evolution and Binding Mechanisms of Intrinsically Disordered Proteins
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Intrinsically disordered proteins (IDPs) make up a considerable fraction of the proteome in eukaryotic organisms. These proteins often act as hubs in interaction networks, harbouring multiple interaction with other proteins, and thus evolution has to walk a tightrope to accommodate new interactions while maintaining the previously established interactions. The ability to accommodate multiple ligands with high specificity is one of the fascinating properties of IDPs, and the molecular details of how this is achieved throughout evolution are largely unknown. The nuclear co-activator binding domain (NCBD) and CREBBP-interacting domain (CID) are intrinsically disordered domains of two transcriptional co-activator proteins. This interaction constitutes one of the earliest examples of a binding reaction where two binding partners fold synergistically upon binding. Previous phylogenetic analysis showed that NCBD is evolutionarily older than CID, which likely emerged after the divergence of the deuterostome and protostome clades of the animal kingdom. When NCBD adapted to the new ligand CID, the affinity of this interaction increased 10-20-fold, while the affinity for some other NCBD ligands were maintained. My thesis work has largely focused around understanding the evolutionary adaptation of the NCBD-CID complex. I have characterised a reconstructed ancestral NCBD-CID complex with respect to structure, folding and binding mechanism and compared these properties to those of the present-day human complex. The results show that the structure and disordered properties of NCBD and CID, as well as their overall binding mechanism, have been moderately conserved throughout evolution. Small differences in the binding mechanism and compactness of the complexes were observed between the most ancestral and present-day human complexes, indicating a somewhat malleable protein-protein interaction that allows for fine-tuning of biophysical properties when new ligands are adopted. Furthermore, I have investigated the impact of disordered regions flanking the binding interface of present-day human CID, using stopped-flow fluorimetry. The disordered regions contributed to an increased affinity to NCBD, although no additional structure was formed upon binding. Ionic strength-dependence curves of the obtained kinetic parameters showed that electrostatic interactions likely do not contribute to the increase in affinity mediated by the disordered flanking regions. These results demonstrate how disordered regions flanking the binding interface regions can contribute to affinity, and highlights the importance of including larger parts of proteins when conducting studies of proteins in vitro.
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| 8. |
- Lama, Dilraj, et al.
(author)
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A druggable conformational switch in the c-MYC transactivation domain
- 2024
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Journal article (peer-reviewed)abstract
- The c-MYC oncogene is activated in over 70% of all human cancers. The intrinsic disorder of the c-MYC transcription factor facilitates molecular interactions that regulate numerous biological pathways, but severely limits efforts to target its function for cancer therapy. Here, we use a reductionist strategy to characterize the dynamic and structural heterogeneity of the c-MYC protein. Using probe-based Molecular Dynamics (MD) simulations and machine learning, we identify a conformational switch in the c-MYC amino-terminal transactivation domain (termed coreMYC) that cycles between a closed, inactive, and an open, active conformation. Using the polyphenol epigallocatechin gallate (EGCG) to modulate the conformational landscape of coreMYC, we show through biophysical and cellular assays that the induction of a closed conformation impedes its interactions with the transformation/transcription domain-associated protein (TRRAP) and the TATA-box binding protein (TBP) which are essential for the transcriptional and oncogenic activities of c-MYC. Together, these findings provide insights into structure-activity relationships of c-MYC, which open avenues towards the development of shape-shifting compounds to target c-MYC as well as other disordered transcription factors for cancer treatment. Here, the authors identify a conformational switch in the amino-terminal transactivation domain of c-MYC, termed coreMYC, which cycles between a closed, inactive state and an open, active conformation. Polyphenol epigallocatechin gallate (EGCG) is used to modulate the conformational landscape of coreMYC, stabilizing the closed and inactive conformation.
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| 9. |
- Lignell, Martin, 1967-
(author)
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Structural Transitions in Helical Peptides : The Influence of Water – Implications for Molecular Recognition and Protein Folding
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition. Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations? Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle. Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone. Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.
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| 10. |
- Lundström, Patrik, et al.
(author)
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Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Ca and side-chain methyl positions in protein
- 2007
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In: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 38:3, s. 199-212
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Journal article (peer-reviewed)abstract
- A simple labeling approach is presented based on protein expression in [1-C-13]- or [2-C-13]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C-alpha sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta 1) are produced with isolated C-13 spins (i.e., no C-13-C-13 one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C-beta positions for 17 of the common 20 amino acids and there are no cases for which C-13(alpha)-(CO)-C-13 spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone N-15 studies. The utility of the labeling is established by recording C-13 R-1 rho and CPMG-based experiments on a number of different protein systems.
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