Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.

Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.

Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Since the matching of SPs/TMs creates the illusion of matching hydrophobic cores, the inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, this work shows explicit examples that the scores of clearly false-positive hits, even in globalmode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, this study finds that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, false positive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. A workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users is provided. While E-value guided extrapolation of protein domain annotation from libraries such as Pfam with the HMMER suite is indispensable for hypothesizing about the function of experimentally uncharacterized protein sequences, it can also complicate the annotation problem. In HMMER2, the E-value is computed from the score via a logistic function or via a domain model-specific extreme value distribution (EVD); the lower of the two is returned as E-value for the domain hit in the query sequence. We demonstrated that, for thousands of domain models, this treatment results in switching from the EVD to the statistical model with the logistic function when scores grow (for Pfam release 23, 99% in the global mode and 75% in the fragment mode). If the score corresponding to the breakpoint results in an E-value above a user-defined threshold (e.g., 0.1), a critical score region with conflicting E-values from the logistic function (below the threshold) and from EVD (above the threshold) does exist. Thus, this switch will affect E-value guided annotation decisions in an automated mode. To emphasize, switching in the fragment mode is of no practical relevance since it occurs only at E-values far below 0.1. Unfortunately, a critical score region does exist for 185 domain models in the hmmpfam and 1748 domain models in the hmmsearch global-search mode. For 145 out the respective 185 models, the critical score region is indeed populated by actual sequences. In total, 24.4% of their hits have a logistic function-derived E-value<0.1 when the EVD provides an E-value>0.1. Examples of false annotations are provided and the appropriateness of a logistic function as alternative to the EVD is critically discussed. This work shows that misguided E-value computation coupled with non-globular regions embedded in domain model library not only causes annotation errors in public databases but also limits the extrapolation power of protein function prediction tasks. So far, the preceding work has demonstrated that sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches from non-polar residues. We found that there are two types of transmembrane helices (TMs) in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intramembrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information. For extending the homologyconcept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments. Theoretical insights from this work were applied to problems of function prediction for specific uncharacterized gene/protein sequences (for example, APMAP and ARXES) and for the functional classification of TM-containing proteins.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 6, 2008). Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 1997. / Includes bibliographical references (leaves 170-181).

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "December 1998" Typescript. Vita. Includes bibliographical references (l. 140-148). Also available on the Internet.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 191-192.

The biosynthesis of integral membrane proteins (IMPs) is an essential cellular process. IMPs comprise roughly 20-30% of the protein coding genes of all organisms, nearly all of which are inserted and assembled at the endoplasmic reticulum (ER). The defining structural feature of IMPs is one or more transmembrane domains (TMDs). TMDs are typically stretches of predominately hydrophobic amino acids that span the lipid bilayer of biological membranes as an alpha helix. TMDs are remarkably diverse in terms of their topological and biophysical properties. In order to accommodate this diversity, the cell has evolved different sets of machinery that cater to particular subsets of proteins. Our knowledge of how the TMDs of IMPs are selectively recognized, chaperoned into the lipid bilayer, and assembled remains incomplete. This thesis is broadly interested in investigating how TMDs are correctly inserted and assembled at the ER. To address this the biosynthesis of multi-pass IMPs was first considered. Multi-pass IMPs contain two to more than twenty TMDs, with TMDs that vary dramatically in terms of their biophysical properties such as hydrophobicity, length, and helical propensity. The beta-1 adrenergic receptor (β1-AR), a member of the G-protein-coupled receptor (GPCR) family was established as a model substrate in an in vitro system where the insertion and folding of its TMDs could be interrogated. Assembly of β1-AR is not a straightforward process, and current models of insertion fail to explain how the known translocation machinery correctly identifies, inserts, and assembles β1-AR TMDs. An in vivo screen in mammalian cells was therefore conducted to identify additional factors which may be important for multi-pass IMP assembly. The ER membrane protein complex (EMC), a well conserved ER-resident complex of unknown biochemical function, was identified as a promising hit potentially involved in this assembly process. The complexity of working with multi-pass IMPs in an in vitro system prompted the investigation of a simpler class of proteins. Tail-anchored proteins (TA) are characterized by a single C-terminal hydrophobic domain that anchors them into membranes. Though structurally simpler compared to multi-pass IMPs, the TMDs of TA proteins are similarly diverse. We found that known TA insertion pathways fail to engage low-to-moderately hydrophobic TMDs. Instead, these are chaperoned in the cytosol by calmodulin (CaM). Transient release from CaM allows substrates to sample the ER, where resident machinery mediates the insertion reaction. The EMC was shown to be necessary for the insertion of these substrates both in vivo and in vitro. Purified EMC in synthetic liposomes catalysed insertion of its TA substrates in a fully reconstituted system to near-native levels. Therefore, the EMC was rigorously established as a TMD insertase. This key functional insight may explain its critical role in the assembly of multi- pass IMPs – which is now amenable to biochemical dissection.

abstract: The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution. This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding &#8764;1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014

Protein quality control is crucial to maintaining cellular function. A failure to clear misfolded, aggregation prone proteins can lead to the accumulation of toxic protein aggregates that interfere with cellular pathways and lead to cell death. In addition, the degradation of partially functional proteins can lead to loss of function diseases. Understanding proteins quality control mechanisms is therefore of fundamental importance to understanding these disease pathways. Systems that operate to monitor the structure of soluble protein domains are now relatively well understood. However, in addition to soluble domains, membrane proteins contain regions that span lipid bilayers, and a key question that remains is where and how these transmembrane domains (TMDs) that fail to assemble correctly or are otherwise aberrant are recognised within subcellular compartments. As such, in this study model chimeric proteins containing the luminal and cytoplasmic domain of the single-spanning membrane protein CD8 and exogenous TMDs derived from polytopic membrane proteins were used to investigate the handling of non-native TMDs in the secretory pathway. CD8 chimeras containing non-native TMDs were found to be recognised by endoplasmic reticulum (ER) quality control pathways. Importantly, ER-associated degradation of CD8 chimeras containing exogenous TMDs was reliant upon ubiquitination of cytoplasmic lysine residues prior to retrotranslocation and dislocation from the ER membrane. In contrast, CD8 containing the endogenous TMD but a misfolded luminal domain could be efficiently degraded when cytoplasmic lysines were removed, suggesting that the retrotranslocation mechanisms for these proteins are distinct and defined by the domain which is misfolded. A proportion of the CD8 chimeras containing non-native TMDs were able to exit the ER, and were retrieved to the ER from the Golgi. Golgi-to-ER retrieval was found to be at least partially mediated by Rer1. CD8 chimeras that escaped ER retrieval could also be retained in the Golgi and subsequently degraded in lysosomes, indicating the presence of an as yet undefined TMD-based Golgi quality control checkpoint in mammalian cells. Furthermore, in contrast to WT CD8 which was stable at the plasma membrane, CD8 chimeras containing non-native TMDs that trafficked to the cell surface were rapidly internalised and sorted to lysosomes. This process was largely independent of the cytoplasmic domain of CD8, suggesting signals within the TMD induced internalisation of these CD8 chimeras. The proportion of the CD8 chimeras that trafficked to the plasma membrane, and the stability of the protein at the cell surface, was dependent upon the presence of polar residues within the TMDs, indicating that exposed polar residues in non-native TMDs may alter the handling of proteins at the Golgi and cell surface. Together, these results further our understanding of the mechanisms by which proteins containing aberrant transmembrane domains are handled at multiple subcellular compartments.