Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access
Subramanian, Kalyanasundaram ; Góra, Artur ; Spruijt, Ruud ; Mitusińska, Karolina ; Suarez-Diez, Maria ; Martins Dos Santos, Vitor ; Schaap, Peter J. - \ 2018
PLoS ONE 13 (2018)6. - ISSN 1932-6203
D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.
Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29
Shabestari, M.H. ; Wolfs, C.J.A.M. ; Spruijt, R.B. ; Amerongen, H. van; Huber, M. - \ 2014
Biophysical Journal 106 (2014)6. - ISSN 0006-3495 - p. 1349 - 1358.
light-harvesting complex - electron-paramagnetic-resonance - comprehensive software package - labeled side-chains - photosystem-ii - distance measurements - conformational-changes - structure prediction - energy-transfer - t4 lysozyme
The structure of the unusually long (~100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows a-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.
Phage Display of Engineered Binding Proteins
Levisson, M. ; Spruijt, R.B. ; Winkel, I.N. ; Kengen, S.W.M. ; Oost, J. van der - \ 2014
Methods in molecular biology 1129 (2014). - ISSN 1064-3745 - p. 211 - 229.
In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.
Simple and rapid quantification of total carotenoids in lyophilized apricots (prunus armeniaca l.) by means of reflectance colorimetry and photoacoustic spectroscopy
Doka, O. ; Ficzek, G. ; Luterotti, S. ; Bicanic, D.D. ; Spruijt, R.B. ; Buijnsters, J. ; Vegvari, G. - \ 2013
Food Technology and Biotechnology 51 (2013)4. - ISSN 1330-9862 - p. 453 - 459.
fruit - vegetables - cultivars - varieties - anthocyanin - pumpkins - maturity - lycopene - vitamin - quality
Photoacoustic spectroscopy (PAS) and reflectance colorimetry are suggested as new tools for the analysis of total carotenoids in lyophilized apricot powders. The data obtained by these two techniques from seven apricot cultivars were compared to those acquired by spectrophotometry and high-performance liquid chromatography (HPLC). Best correlations were found between the total carotenoid (TC) content (obtained by VIS spectrophotometry: 1.2-3.4 mg per 100 g of fresh mass) and colorimetric index a* (a* represents the redness of the investigated sample), as well as either argon-ion laser- or xenon-lamp-based PAS. In all three cases linear correlations were comparable. However, according to the sensitivity and precision data, expressed via limit of detection (LOD) and measurement repeatability the Xe-lamp-based PAS is a preferred approach, followed by colorimetric index a* and Ar-ion laser PAS. Both PAS methods exhibit practically the same Pearson's correlation coefficient (R=0.987 and R=0.991) values. Nevertheless, residual sum of squares (RSS) and residual standard deviation of the linear regression (s(y/x)) differ markedly For Xe-lamp-based PAS these parameters were much lower than in the case of Ar-ion laser PAS. Likewise, analysis imprecision amounted to relative standard deviation (RSD) of 1-3 % for Xe-lamp PAS and 2-6 % for Ar-ion laser PAS. On the other hand, as expected, the calibration sensitivity achieved for the PAS signal induced by an Ar-ion laser at 481 nm was substantially higher than that of a Xe-lamp at 470 nm. Nevertheless, according to much lower sy/x, the corresponding LOD for Xe-lamp PAS was still two times lower than that of Ar-ion-based laser PAS (0.59 vs. 1.10 mg per 100 g). Unlike this, Ar-ion laser PAS showed more favourable instrumental precision and standard error of the weighed mean when compared to the Xe-lamp PAS (0.1-0.6 and 0.1-0.3 % vs. 0.5-8.0 and 0.4-1.7 %, respectively). As far as colorimetric indices are concerned, only a* proved to be analytically useful; excellent R but rather modest RSS and s(y/x) resulted in LOD value of 0.70 mg per 100 g and acceptable analysis imprecision of up to 3 %. The outcome of this research provides sufficient amount of evidence that analytical methods such as reflectance colorimetry and PAS without the use of any chemicals are feasible for reliable quantification of total carotenoids in freeze-dried apricot homogenates.
Direct photothermal techniques for quantification of anthocyanins in sour cherry cultivars
Doka, O. ; Ficzek, G. ; Bicanic, D.D. ; Spruijt, R.B. ; Luterotti, S. ; Toth, M. ; Buijnsters, J.G. ; György Végvári, G. - \ 2011
Talanta 84 (2011)2. - ISSN 0039-9140 - p. 341 - 346.
cancer prevention - prunus-cerasus - tart cherry - l. - antioxidant - flavonoids - extracts - rat - ldl
The analytical performance of the newly proposed laser-based photoacoustic spectroscopy (PAS) and of optothermal window (OW) method for quantification of total anthocyanin concentration (TAC) in five sour cherry varieties is compared to that of the spectrophotometry (SP). High performance liquid chromatography (HPLC) was used to identify and quantify specific anthocyanins. Both, PAS and OW are direct methods that unlike SP and HPLC obviate the need for the extraction of analyte. The outcome of the study leads to the conclusion that PAS and OW are both suitable for quick screening of TAC in sour cherries. The correlation between the two methods and SP is linear with R2 = 0.9887 for PAS and R2 = 0.9918 for OW, respectively. Both methods are capable of the rapid determination of TAC in sour cherries without a need for a laborious sample pretreatment.
Assaying total carotenoids in flours of corn and sweet potato flours by laser photoacoustic spectroscopy
Luterotti, S. ; Bicanic, D.D. ; Kijak, K. ; Grbesa, D. ; Martinez, E. ; Spruijt, R.B. - \ 2011
Food Biophysics 6 (2011)1. - ISSN 1557-1858 - p. 12 - 19.
thermal-diffusivity - particle-size - beta-carotene - stability - lycopene
This study describes the application of the laser photoacoustic spectroscopy (PAS) for quantification of total carotenoids (TC) in corn flours and sweetpotato flours. Overall, thirty-three different corn flours and nine sweetpotato flours were investigated. All PAS measurements were performed at room temperature using 488-nm argon laser radiation for excitation and mechanical modulation of 9 and 30 Hz. The measurements were repeated within a run and within several days or months. The UV–Vis spectrophotometry was used as the reference method. The concentration range that allows for the reliable analysis of TC spans a region from 1 to 40 mg kg-1 for corn flours and from 9 to 40 mg kg-1 for sweetpotato flours. In the case of sweetpotato flours, the quantification may extend even to 240 mg kg-1 TC. The estimated detection limit values for TC in corn and sweetpotato flours were 0.1 and 0.3 mg kg-1, respectively. The computed repeatability (n¿=¿3–12) and intermediate precision (n¿=¿6–28) RSD values at 9 and 30 Hz are comparable: 0.1–17.1% and 5.3–14.7% for corn flours as compared with 1.4–9.1% and 4.2–23.0% for sweetpotato flours. Our results show that PAS can be successfully used as a new analytical tool to simply and rapidly screen the flours for their nutritional potential based on the total carotenoid concentration
Exploiting direct and indirect methods for the detection of the total carotenoid content in dried pasta
Doka, O. ; Bicanic, D.D. ; Végvári, G. ; Buijnsters, J.G. ; Spruijt, R.B. ; Luterotti, S. - \ 2010
European Food Research and Technology 230 (2010)6. - ISSN 1438-2377 - p. 813 - 819.
performance liquid-chromatography - egg-yolk - beta-carotene - wheat - quantification - flour - identification - vegetables - yellow - fruits
The total carotenoid concentration (TCC) of several commercially available dried pastas prepared with or without eggs was assessed by means of the two well-established destructive approaches [spectrophotometry (SP) and high-performance liquid chromatography (HPLC)] and three non-destructive, direct (i.e., no sample preparation required) methods (resonance Raman spectroscopy, photoacoustic (PA) spectroscopy and colorimetry). The results obtained by the three direct methods correlate well with the TCC of investigated dried pastas assessed by SP and HPLC. The best linear correlation and detection limit were achieved for the PA method at 470 nm
Viruses: incredible nanomachines. New advances with filamentous phages
Hemminga, M.A. ; Vos, W.L. ; Nazarov, P.V. ; Koehorst, R.B.M. ; Wolfs, C.J.A.M. ; Spruijt, R.B. ; Stopar, D. - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 541 - 550.
major coat protein - transmembrane alpha-helix - membrane-protein - bacteriophage m13 - nmr-spectroscopy - ff fd - site - dynamics - display - domain
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 accou
Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy
Berghuis, B.A. ; Spruijt, R.B. ; Koehorst, R.B.M. ; Hoek, A. van; Laptenok, S. ; Oort, B.F. van; Amerongen, H. van - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 631 - 638.
light-harvesting complex - radical-cation formation - plant antenna protein - energy-transfer - photosystem-ii - green plants - escherichia-coli - refractive-index - chlorophyll-a - pump-probe
A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (~20–70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (~15 Å), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model
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
Koehorst, R.B.M. ; Laptenok, S. ; Oort, B.F. van; Hoek, A. van; Spruijt, R.B. ; Stokkum, I.H.M. van; Amerongen, H. van; Hemminga, M.A. - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 647 - 656.
major coat protein - charge-transfer fluorescence - n-terminal domain - hydration dynamics - excited-state - prodan - m13 - solvation - laurdan - relaxation
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.
Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness
Stopar, D. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2009
Biochimica et Biophysica Acta. Biomembranes 1788 (2009)10. - ISSN 0005-2736 - p. 2217 - 2221.
membrane-proteins - tryptophan residues - amino-acids - peptides - topology - helix - spectroscopy - orientation
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 ( 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
Membrane protein frustration: protein incorporation into hydrophobic mismatched binary lipid mixtures
Stopar, D. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2009
Biophysical Journal 96 (2009)4. - ISSN 0006-3495 - p. 1408 - 1414.
major coat protein - bacteriophage m13 - phase-transitions - acyl-chain - bilayers - fluid - phosphatidylcholines - spectroscopy - domain - solubilization
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 ¿-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
Site-directed spin labeling study of the light-harvesting complex CP29
Kavalenka, A.A. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Strancar, J. ; Croce, R. ; Hemminga, M.A. ; Amerongen, H. van - \ 2009
Biophysical Journal 96 (2009)9. - ISSN 0006-3495 - p. 3620 - 3628.
chlorophyll-a/b protein - photosystem-ii subunit - plant antenna protein - n-terminal domain - energy-transfer - conformational-changes - biosystem complexity - membrane-proteins - escherichia-coli - binding protein
The topology of the long N-terminal domain (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
Vos, W.L. ; Nazarov, P.V. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2009
Trends in Biochemical Sciences 34 (2009)5. - ISSN 0968-0004 - p. 249 - 255.
major coat protein - amino-acids - filamentous bacteriophages - nmr-spectroscopy - fd - dynamics - domain - helix - environments - resolution
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 ¿-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
Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29
Oort, B.F. van; Murali, S. ; Wientjes, E. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hoek, A. van; Croce, R. ; Amerongen, H. van - \ 2009
Chemical Physics 357 (2009)1-3. - ISSN 0301-0104 - p. 113 - 119.
light-harvesting complexes - photosystem-ii subunit - chlorophyll a/b complex - plant antenna protein - green plants - absorption properties - escherichia-coli - refractive-index - excited-states - pump-probe
The photosynthetic minor antenna complex CP29 of higher plants was singly mutated, overexpressed in Escherichia coli, selectively labeled with the fluorescent dye TAMRA at three positions in the N-terminal domain, and reconstituted with its natural pigments. Picosecond fluorescence experiments revealed rapid excitation energy transfer (20 ps) from TAMRA covalently attached to a cysteine at either position 4 or 97 (near the beginning and end of the N-terminal domain) to the chlorophylls in the hydrophobic part of the protein. This indicates that the N-terminus is folded back on the hydrophobic core. In 20% of the complexes, efficient transfer was lacking, indicating that the N-terminus can adopt different conformations. Time-resolved polarized fluorescence measurements demonstrate that the non-transferring conformations only allow restricted rotational motion of the dye molecule. When TAMRA was attached to a cysteine at position 40, the overall transfer efficiency was far lower, reflecting a larger distance to the hydrophobic region
|Direct detection of beta carotene in the model mixtures of pre-cooked white corn flour: the multi methods approach
Bicanic, D.D. ; Spruijt, R.B. ; Doka, O. ; Krzton, A. ; Oomens, J. ; Miller, G. ; Jalink, H. ; Billinghurst, B. ; Michaelian, K. ; Koehorst, R.B.M. ; Persijn, S. ; Schoor, R. van der; Samkovic, K. ; Boban, M. ; Kurtanjek, Z. ; Rendevski, S. ; Goracinova, K. ; Hulshof, P.J.M. ; Roekel-Jansen, G.C. van; Golic-Baric, I. ; Zijl, A. van; Harbinson, J. - \ 2008
In: Proceedings of the The 6th Croatian Congress of Food Technologists, Biotechnologists and Nutritionists. - CEFood - p. 610 - 625.
Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment
Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2008
Biophysical Journal 94 (2008). - ISSN 0006-3495 - p. 3945 - 3955.
major coat protein - charge-transfer fluorescence - excited-state - phospholipid-bilayers - transmembrane domain - laurdan fluorescence - solvent relaxation - bacteriophage m13 - prodan - water
We present a new and simple method based on site-directed fluorescence labeling using the BADAN label that allows to examine protein-lipid interactions in great detail. We apply this approach to a membrane-embedded mainly -helical reference protein, the M13 major coat protein, of which in a high-throughput approach 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra are analyzed using a three-component spectral model that enables to separate Stokes shift contributions from water and internal label dynamics, and protein topology. It is found that most of the fluorescence originates from BADAN labels that are hydrogen bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method reveals 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 a hydration level and local polarity, given by the local dielectric constant
Structure of membrane embedded M13 major coat protein is insensitive to hydrophobic stress
Vos, W.L. ; Schor, M. ; Nazarov, P.V. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2007
Biophysical Journal 93 (2007)10. - ISSN 0006-3495 - p. 3541 - 3547.
lipid-bilayer - molecular-dynamics - alpha-helices - model - mismatch - conformation - modulation - fret - aggregation - association
The structure of a membrane-embedded -helical reference protein, the M13 major coat protein, is characterized under different conditions of hydrophobic mismatch using fluorescence resonance energy transfer in combination with high-throughput mutagenesis. We show that the structure is similar in both thin (14:1) and thick (20:1) phospholipid bilayers, indicating that the protein does not undergo large structural rearrangements in response to conditions of hydrophobic mismatch. We introduce a "helical fingerprint" analysis, showing that amino acid residues 1¿9 are unstructured in both phospholipid bilayers. Our findings indicate the presence of -helical domains in the transmembrane segment of the protein; however, no evidence is found for a structural adaptation to the degree of hydrophobic mismatch. In light of current literature, and based on our data, we conclude that aggregation (at high protein concentration) and adjustment of the tilt angle and the lipid structure are the dominant responses to conditions of hydrophobic mismatch.
Decomposition of ESR spectra using MALDI-TOF mass spectrometry
Vos, W.L. ; Vermeer, L.S. ; Wolfs, C. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2006
Analytical Chemistry 78 (2006)15. - ISSN 0003-2700 - p. 5296 - 5301.
electron-paramagnetic-resonance - major coat protein - spin labels - containing peptides - lipid-bilayer - tryptophan - distances - helix - ions
ESR ( or EPR) spectroscopy on spin-labeled site-directed cysteine mutants is ideally suited for structural studies of membrane proteins due to its high sensitivity and its low demands with respect to sample purity and preparation. Many features can be inferred from the spectral line shape of an ESR spectrum, but the analysis of ESR spectra is complicated when multiple sites with different line shapes are present. Here, we present a method to decompose the spectrum of a doubly labeled peptide that is composed of a singly labeled, noninteracting component and a doubly labeled, dipolar-broadened component using a combination of optical and matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry. The effect on the interspin distance calculation based on the dipolar broadening is quantified and discussed.
Anchoring mechanisms of membrane-associated M13 major coat protein
Stopar, D. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2006
Chemistry and Physics of Lipids 141 (2006)1-2. - ISSN 0009-3084 - p. 83 - 93.
water interface - lipid-bilayers - nmr-spectroscopy - transmembrane domain - tryptophan residues - backbone dynamics - aromatic residues - bacteriophage m13 - solid-state - model
Bacteriophage M13 major coat protein is extensively used as a biophysical, biochemical, and molecular biology reference system for studying membrane proteins. The protein has several elements that control its position and orientation in a lipid bilayer. The N-terminus is dominated by the presence of negatively charged amino acid residues (Glu2, Asp4, and Asp5), which will always try to extend into the aqueous phase and therefore act as a hydrophilic anchor. The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains (Phe 11, Tyr21, Tyr24, Trp26, Phe42, Phe45, Lys40, Lys43, and Lys44) that fine-tune the association of the protein to the lipid bilayer. The interfacial Tyr residues are important recognition elements for precise protein positioning, a function that cannot be performed optimally by residues with an aliphatic character. The Trp26 anchor is not very strong: depending on the context, the tryptophan residue may move in or out of the membrane. On the other hand, Lys residues and Phe residues at the C-terminus of the protein act in a unique concerted action to strongly anchor the protein in the lipid bilayer.