Biophysical Chemistry in Groningen - A Personal Note.

This paper describes the start of the Biophysical Chemistry group of prof. Herman J.C. Berendsen (1934-2019) in Groningen, The Netherlands in the years from 1964 to 1974.

Distance measurements on orthogonally spin-labeled membrane spanning WALP23 polypeptides.

EPR-based Gd(III)-nitroxide distance measurements were performed on a series of membrane-incorporated orthogonally labeled WALP23 polypeptides. The obtained distance distributions were stable upon the change of detection frequency from 10 GHz (X-band) to 35 GHz (Q-band). The α-helical pitch of WALP23 polypeptide could be experimentally observed, despite the flexibility of the two spin labels. The spectroscopic properties of Gd(III) ions and nitroxide radicals allow detecting both types of paramagnetic species selectively in different EPR experiments. In particular, this spectroscopic selectivity allows for supplementing Gd(III)-nitroxide distance measurements with independent checks of polypeptide aggregation and with measurements of the local environment of the nitroxide spin labels. All mentioned additional checks do not require preparation of further samples, as it is the case in the experiments with pairs of identical nitroxide spin labels.

Multiple Pathway Relaxation Enhancement in the System Composed of Three Paramagnetic Species: Nitroxide Radical-Ln(3+)-O2.

Longitudinal relaxation of nitroxide spin-labels has been measured for a membrane-incorporated α-helical polypeptide in the presence and absence of residual amounts of membrane-dissolved O2 and paramagnetic Dy(3+) ions. Such a model system, containing three different types of paramagnetic species, provides an important example of nonadditivity of two different relaxation channels for the nitroxide spins.

Profiling of dynamics in protein-lipid-water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths.

Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein-lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane-water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains.

Viruses: incredible nanomachines. New advances with filamentous phages.

During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account.

Conformational studies of peptides representing a segment of TM7 from H+-VO-ATPase in SDS micelles.

The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an alpha-helical conformation or aggregated beta-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.e., MTM7). It is concluded that the bulky, positively charged arginine residue located in the center of both peptides has a destabilizing effect on the helical conformation of the SDS-solubilized peptides, leading to beta-sheet formation and subsequent aggregation.

Molecular dynamics simulations reveal that AEDANS is an inert fluorescent probe for the study of membrane proteins.

Computer simulations were carried out of a number of AEDANS-labeled single cysteine mutants of a small reference membrane protein, M13 major coat protein, covering 60% of its primary sequence. M13 major coat protein is a single membrane-spanning, alpha-helical membrane protein with a relatively large water-exposed region in the N-terminus. In 10-ns molecular dynamics simulations, we analyze the behavior of the AEDANS label and the native tryptophan, which were used as acceptor and donor in previous FRET experiments. The results indicate that AEDANS is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein.

SDSL-ESR-based protein structure characterization.

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.

Tilt and rotation angles of a transmembrane model peptide as studied by fluorescence spectroscopy.

In this study the membrane orientation of a tryptophan-flanked model peptide, WALP23, was determined by using peptides that were labeled at different positions along the sequence with the environmentally sensitive fluorescent label BADAN. The fluorescence properties, reflecting the local polarity, were used to determine the tilt and rotation angles of the peptide based on an ideal alpha-helix model. For WALP23 inserted in dioleoylphosphatidylcholine (DOPC), an estimated tilt angle of the helix with respect to the bilayer normal of 24 degrees +/- 5 degrees was obtained. When the peptides were inserted into bilayers with different acyl chain lengths or containing different concentrations of cholesterol, small changes in tilt angle were observed as response to hydrophobic mismatch, whereas the rotation angle appeared to be independent of lipid composition. In all cases, the tilt angles were significantly larger than those previously determined from (2)H NMR experiments, supporting recent suggestions that the relatively long timescale of (2)H NMR measurements may result in an underestimation of tilt angles due to partial motional averaging. It is concluded that although the fluorescence technique has a rather low resolution and limited accuracy, it can be used to resolve the discrepancies observed between previous (2)H NMR experiments and molecular-dynamics simulations.

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness.

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm(-1) on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less ( approximately 70 cm(-1)). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.

Site-directed spin-labeling study of the light-harvesting complex CP29.

The topology of the long N-terminal domain (approximately 100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.

From 'I' to 'L' and back again: the odyssey of membrane-bound M13 protein.

The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped alpha-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein. Recently, site-directed labelling approaches have enabled the study of the coat protein under conditions that more closely mimic the in vivo membrane-bound state. Interestingly, the structure that has emerged from this work is I-shaped and similar to the structure in the phage-bound state.

Structural properties of a peptide derived from H+ -V-ATPase subunit a.

The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H(+)-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The helical region extends well beyond A738, as was previously suggested based on NMR studies of a similar peptide in DMSO. The pKa of both histidine residues that are important for proton transport was measured in water and in SDS. The differences that are found demonstrate that the histidine residues interact with the SDS polar heads. In detergent, circular dichroism data indicate that the secondary structure of the peptide depends on the pH and the type of detergent used. Using solid-state NMR, it is shown that the peptide is immobile in phospholipid bilayers, which means that it is probably not a single transmembrane helix in these samples. The environment is important for the structure of TM7, so in subunit a it is probably held in place by the other transmembrane helices of this subunit.

Membrane protein frustration: protein incorporation into hydrophobic mismatched binary lipid mixtures.

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall alpha-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.

Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy.

Site-directed spin-labeling electron spin resonance (SDSL-ESR) is a promising tool for membrane protein structure determination. Here we propose a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. We test the sensitivity of this approach for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels employing high-throughput site-directed mutagenesis. We find a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to the one determined by fluorescence resonance energy transfer analysis [P.V. Nazarov, R.B.M. Koehorst, W.L. Vos, V.V. Apanasovich, M.A. Hemminga, FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein, Biophys. J. 92 (2007) 1296-1305].

An N-terminal diacidic motif is required for the trafficking of maize aquaporins ZmPIP2;4 and ZmPIP2;5 to the plasma membrane.

Maize plasma membrane aquaporins (ZmPIPs, where PIP is the plasma membrane intrinsic protein) fall into two groups, ZmPIP1s and ZmPIP2s, which, when expressed alone in mesophyll protoplasts, are found in different subcellular locations. Whereas ZmPIP1s are retained in the endoplasmic reticulum (ER), ZmPIP2s are found in the plasma membrane (PM). We previously showed that, when co-expressed with ZmPIP2s, ZmPIP1s are relocalized to the PM, and that this relocalization results from the formation of hetero-oligomers between ZmPIP1s and ZmPIP2s. To determine the domains responsible for the ER retention and PM localization, respectively, of ZmPIP1s and ZmPIP2s, truncated and mutated ZmPIPs were generated, together with chimeric proteins created by swapping the N- or C-terminal regions of ZmPIP2s and ZmPIP1s. These mutated proteins were fused to the mYFP and/or mCFP, and the fusion proteins were expressed in maize mesophyll protoplasts, and were then localized by microscopy. This allowed us to identify a diacidic motif, DIE (Asp-Ile-Glu), at position 4-6 of the N-terminus of ZmPIP2;5, that is essential for ER export. This motif was conserved and functional in ZmPIP2;4, but was absent in ZmPIP2;1. In addition, we showed that the N-terminus of ZmPIP2;5 was not sufficient to cause the export of ZmPIP1;2 from the ER. A study of ZmPIP1;2 mutants suggested that the N- and C-termini of this protein are probably not involved in ER retention. Together, these results show that the trafficking of maize PM aquaporins is differentially regulated depending on the isoform, and involves a specific signal and mechanism.

Molecular dynamics study of the solvation of an alpha-helical transmembrane peptide by DMSO.

A 10-ns molecular dynamics study of the solvation of a hydrophobic transmembrane helical peptide in dimethyl sulfoxide (DMSO) is presented. The objective is to analyze how this aprotic polar solvent is able to solvate three groups of amino acid residues (i.e., polar, apolar, and charged) that are located in a stable helical region of a transmembrane peptide. The 25-residue peptide (sMTM7) used mimics the cytoplasmic proton hemichannel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The three-dimensional structure of peptide sMTM7 in DMSO has been previously solved by NMR spectroscopy. The radial and spatial distributions of the DMSO molecules surrounding the peptide as well as the number of hydrogen bonds between DMSO and the side chains of the amino acid residues involved are extracted from the molecular dynamics simulations. Analysis of the molecular dynamics trajectories shows that the amino acid side chains are fully embedded in DMSO. Polar and positively charged amino acid side chains have dipole-dipole interactions with the oxygen atom of DMSO and form hydrogen bonds. Apolar residues become solvated by DMSO through the formation of a hydrophobic pocket in which the methyl groups of DMSO are pointing toward the hydrophobic side chains of the residues involved. The dual solvation properties of DMSO cause it to be a good membrane-mimicking solvent for transmembrane peptides that do not unfold due to the presence of DMSO.

Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment.

The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly alpha-helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.

Solubilization of V-ATPase transmembrane peptides by amphipol A8-35.

Two transmembrane peptides encompassing the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae were studied as complexes with APols A8-35 by CD and fluorescence spectroscopy, with the goal to use APols to provide a membrane-mimicking environment for the peptides. CD spectroscopy was used to obtain the overall secondary structure of the peptides, whereas fluorescence spectroscopy provided information about the local environment of their tryptophan residues. The fluorescence results indicate that both peptides are trapped by APols and the CD results that they adopt a beta-sheet conformation. This result is in contrast with previous work that showed that the same peptides are alpha-helical in SDS micelles and organic solvents. These observations are discussed in the context of APol physical-chemical properties and transmembrane peptide structural propensity.

Membrane-bound peptides from V-ATPase subunit a do not interact with an indole-type inhibitor.

The V-ATPases are ATP-dependent proton pumps, found in virtually all cells, responsible for acidification of organelles and energizing of plasma membranes. Its role in diseases, such as osteoporosis and metastatic cancer, makes the V-ATPase a potential drug target. Short synthetic peptides that are presented here mimic the 7th transmembrane domain (TM7) of subunit a (Vph1p) of Saccharomyces cerevisiae V-ATPase, an essential part of the membrane-bound VO domain, where proton translocation takes place. The peptides adopt a transmembrane configuration only in membranes containing anionic lipids, stressing the importance of strong interfacial anchoring by the flanking lysines. Peptide P1, which contains the essential arginine R735, is monomeric, whereas peptide P2, which lacks this extra charge, tends to aggregate in the membrane. SB 242784, which is a highly potent inhibitor of V-ATPase, does not show any interaction with the peptides, indicating that TM7 alone is not sufficient for inhibitor binding.